Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes

ABSTRACT

Compositions and methods for reducing the level of nornicotine and N′-nitrosonornicotine (NNN) in Nicotiana plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for cytochrome P450s that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Expression cassettes, vectors, plants, and plant parts thereof comprising inhibitory sequences that target expression or function of the disclosed cytochrome P450 polypeptides are also provided. Methods for the use of these novel sequences to inhibit expression or function of cytochrome P450 polypeptides involved in this metabolic conversion are also provided. The methods find use in the production of tobacco products that have reduced levels of nornicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 15/883,931, filed Jan. 30, 2018 (allowed), which isa continuation of U.S. patent application Ser. No. 14/878,083, filedOct. 8, 2015 (now U.S. Pat. No. 9,913,451), which is a continuation ofU.S. patent application Ser. No. 12/971,801, filed Dec. 17, 2010 (nowU.S. Pat. No. 9,187,759), which is a continuation of U.S. applicationSer. No. 11/580,765, filed Oct. 13, 2006 (now U.S. Pat. No. 7,884,263),which is a continuation-in-part of International Application No.PCT/US2005/005665, filed Feb. 23, 2005, the contents of which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to compositions and methods for reducing the levelof nornicotine and its metabolite, N′-nitrosonornicotine, in a plantthat is a member of the genus Nicotiana, particularly compositions andmethods for inhibiting expression or function of a cytochrome P450polypeptide involved in the metabolic conversion of nicotine tonornicotine.

BACKGROUND OF THE INVENTION

The predominant alkaloid found in commercial tobacco varieties isnicotine, typically accounting for 90-95% of the total alkaloid pool.The remaining alkaloid fraction is comprised primarily of threeadditional pyridine alkaloids: nornicotine, anabasine, and anatabine.Nornicotine is generated directly from nicotine through the activity ofthe enzyme nicotine N-demethylase (FIG. 1). Nornicotine usuallyrepresents less than 5% of the total pyridine alkaloid pool, but througha process termed “conversion,” tobacco plants that initially producevery low amounts of nornicotine give rise to progeny that metabolically“convert” a large percentage of leaf nicotine to nornicotine. In tobaccoplants that have genetically converted (termed “converters”), the greatmajority of nornicotine production occurs during the senescence andcuring of the mature leaf (Wernsman and Matzinger (1968) Tob. Sci.12:226-228). Burley tobaccos are particularly prone to geneticconversion, with rates as high as 20% per generation observed in somecultivars.

During the curing and processing of the tobacco leaf, a portion of thenornicotine is metabolized to the compound N′-nitrosonornicotine (NNN;FIG. 1), a tobacco-specific nitrosamine (TSNA) that has been shown to becarcinogenic in laboratory animals (Hecht and Hoffmann (1990) CancerSurveys 8:273-294; Hoffmann et al. (1994) J. Toxicol. Environ. Health41:1-52; Hecht (1998) Chem. Res. Toxicol. 11:559-603). In flue-curedtobaccos, TSNAs were found to be predominantly formed through thereaction of alkaloids with the minute amounts of nitrogen oxides presentin combustion gases formed by the direct-fired heating systems found intraditional curing barns (Peele and Gentry (1999) “Formation ofTobacco-specific Nitrosamines in Flue-cured Tobacco,” CORESTA Meeting,Agro-Phyto Groups, Suzhou, China). Retrofitting these curing barns withheat-exchangers virtually eliminated the mixing of combustion gases withthe curing air and dramatically reduced the formation of TSNAs intobaccos cured in this manner (Boyette and Hamm (2001) Rec. Adv. Tob.Sci. 27:17-22.). In contrast, in the air-cured Burley tobaccos, TSNAformation proceeds primarily through reaction of tobacco alkaloids withnitrite, a process catalyzed by leaf-borne microbes (Bush et al. (2001)Rec. Adv. Tob. Sci. 27:23-46). Thus far, attempts to reduce TNSAsthrough modification of curing conditions while maintaining acceptablequality standards have not proven to be successful for the air-curedtobaccos.

In Burley tobaccos, a positive correlation has been found between thenornicotine content of the leaf and the amount of NNN that accumulatesin the cured product (Bush et al. (2001) Rec. Adv. Tob. Sci. 27:23-46;Shi et al. (2000) Tob. Chem. Res. Conf. 54:Abstract 27). However,keeping nornicotine levels at a minimum has been difficult because ofthe conversion phenomenon that results in a continual introduction ofhigh nornicotine-producing plants within commercially grown Burleypopulations. Minimizing the number of Burley plants that accumulate highlevels of nornicotine has traditionally been the responsibility of plantbreeders and seed producers. Though the percentage of converter plantsthat are ultimately grown in farmers' fields can be reduced through theroguing of converter plants during the propagation of seed stocks, thisprocess is costly, time-consuming, and imperfect.

Previous studies have shown that once a plant has converted, the highnornicotine trait is inherited as a single dominant gene (Griffith etal. (1955) Science 121:343-344; Burk and Jeffrey (1958) Tob. Sci.2:139-141; Mann et al. (1964) Crop Sci. 4:349-353). The nature of thisgene, however, is currently unknown. In the most simple of scenarios,the conversion locus may represent a nonfunctional nicotineN-demethylase gene that regains its function in converter plants,possibly through the mobilization of a mutation-inducing transposableelement. Alternatively, the converter locus may encode a protein thatinitiates a cascade of events that ultimately enables the plant tometabolize nicotine to nornicotine, which would mean that multiple genesmay be involved.

Regardless of whether there are one or many genes associated with theconversion process, it is clear that the gene(s) encoding polypeptideshaving nicotine demethylase activity play a pivotal role in thisprocess. Although the inability to purify active nicotine N-demethylasefrom crude extracts has impeded the isolation and identification of thisenzyme, there is some evidence that a member of the cytochrome P450superfamily of monooxygenases may be involved (Hao and Yeoman (1996)Phytochem. 41:477-482; Hao and Yeoman (1996) Phytochem. 42:325-329;Chelvarajan et al. (1993) J. Agric. Food Chem. 41:858-862; Hao andYeoman (1998)J. Plant Physiol. 152:420-426). However, these studies arenot conclusive, as the classic P450 inhibitors carbon monoxide andtetcylasis have failed to lower enzyme activity at rates comparable toother reported P450-mediated reactions (Chelvarajan et al. (1993) J.Agric. Food Chem. 41:858-862).

Furthermore, the cytochrome P450s are ubiquitous, transmembrane proteinsthat participate in the metabolism of a wide range of compounds(reviewed by Schuler (1996) Crit. Rev. Plant Sci. 15:235-284; Schulerand Werck-Reichhart (2003) Annu. Rev. Plant Biol. 54:629-667). Examplesof biochemical reactions mediated by cytochrome P450s includehydroxylations, demethylations, and epoxidations. In plants, thecytochrome P450 gene families are very large. For example, total genomesequence examination has revealed 272 predicted cytochrome P450 genes inArabidopsis and at least 455 unique cytochrome P450 genes in rice (see,for example, Nelson et al. (2004) Plant Physiol. 135(2):756-772). Eventhough cytochrome P450 has been implicated as having a role in themetabolic conversion of nicotine to nornicotine, identification of keyparticipating members of this protein family remains a challenge.

Aside from serving as a precursor for NNN, recent studies suggest thatthe nornicotine found in tobacco products may have additionalundesirable health consequences. Dickerson and Janda demonstrated thatnornicotine causes aberrant protein glycation within the cell (Dickersonand Janda (2002) Proc. Natl. Acad. Sci. USA 99:15084-15088).Concentrations of nornicotine-modified proteins were found to be muchhigher in the plasma of smokers compared to nonsmokers. Furthermore,this same study showed that nornicotine can covalently modify commonlyprescribed steroid drugs such as prednisone. Such modifications have thepotential of altering both the efficacy and toxicity of these drugs.

In view of the difficulties associated with conversion and theundesirable health effects of nornicotine accumulation, improved methodsfor reducing the nornicotine content in tobacco varieties, particularlyBurley tobacco, are therefore desirable. Such methods would not onlyhelp ameliorate the potential negative health consequences of thenornicotine per se as described above, but should also concomitantlyreduce NNN levels.

SUMMARY OF THE INVENTION

Compositions and methods for reducing the nornicotine content in plantsthat are members of the genus Nicotiana are provided. Compositionsinclude isolated cytochrome P450 polynucleotides and polypeptides thatare involved in the metabolic conversion of nicotine to nornicotine inplants, particularly Nicotiana species. The isolated polynucleotidescomprise the nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9,or 11, a nucleotide sequence encoding a polypeptide as set forth in SEQID NO:2, 4, 6, 8, 10, or 12, and fragments and variants thereof.Isolated polypeptides of the invention comprise an amino acid sequenceset forth in SEQ ID NO:2, 4, 6, 8, 10, or 12, an amino acid sequenceencoded by the nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9,or 11, and fragments and variants thereof.

The polynucleotides of the invention find use in suppressing expressionof a cytochrome P450 that is involved in the metabolic conversion ofnicotine to nornicotine in a plant, including the cytochrome P450s ofthe present invention. In this manner, compositions further includeexpression cassettes comprising an inhibitory sequence that is capableof inhibiting expression or function of a cytochrome P450 polypeptide ofthe invention, where the inhibitory sequence is operably linked to apromoter that is functional in a plant cell. In some embodiments, theinhibitory sequence comprises the sequence set forth in SEQ ID NO:1, 3,5, 7, 9, 11, 13, 14, 15, or 16, or a complement or fragment thereof.Compositions also include transformed plants and plant parts thatcomprise an expression cassette of the present invention, optionallystably incorporated into the genome of the plant. Further provided aretobacco products, including chewing tobacco, snuff, cigarettes, pipetobacco, and cigars, having a reduced level of nornicotine, and itsrelated nitrosamine, N′-nitrosonornicotine.

The methods of the invention comprise inhibiting the expression orfunction of a cytochrome P450 polypeptide of the present invention. Insome embodiments, an expression cassette comprising an inhibitorysequence that targets expression or function of a cytochrome P450polypeptide of the present invention is introduced into the plant orplant part of interest, wherein expression of the inhibitory sequenceproduces a polynucleotide or polypeptide that inhibits expression orfunction of a cytochrome P450 polypeptide of the invention. In one suchembodiment, the inhibitory sequence comprises a sequence set forth inSEQ ID NO:1, 3, 5, 7, 9, 11, 13, 14, 15, or 16, or a complement orfragment thereof.

The methods of the invention find use in the production of Nicotianaplants that have decreased levels of nornicotine and its metabolite, thenitrosamine N′-nitrosonornicotine, within the leaf and stem tissues.When harvested, the leaf and stem tissues of these plants can beutilized to produce tobacco products having reduced levels ofnornicotine and this tobacco-specific nitrosamine, and thus reducedcarcinogenic potential for individuals consuming these products orexposed to secondary smoke derived from these products.

Also provided are transgenic Nicotiana plants having a nicotine tonornicotine conversion rate of less than about 2%, where the plantscomprise a heterologous nucleic acid construct comprising a promotercapable of functioning in a plant cell operably linked to a nucleic acidsequence having a first nucleotide sequence comprising a fragment ofbetween about 100 nucleotides and about 400 nucleotides of a Nicotiananicotine demethylase polynucleotide and a second nucleotide sequencecapable of forming a double-stranded RNA with the first nucleotidesequence, where the transgenic Nicotiana plants are transgenic converterlines of Nicotiana. In some embodiments, the Nicotiana nicotinedemethylase polynucleotide is a tobacco nicotine demethylasepolynucleotide.

The present invention also provides a recombinant nucleic acid constructcomprising a promoter capable of functioning in a plant cell operablylinked to a nucleic acid sequence having a first nucleotide sequencecomprising a fragment of between about 100 nucleotides and about 400nucleotides of a tobacco nicotine demethylase polynucleotide and asecond nucleotide sequence capable of forming a double-stranded RNA withthe first nucleotide sequence.

Also provided are methods of reducing the conversion of nicotine tonornicotine in a Nicotiana plant comprising transforming a Nicotianaplant with a recombinant nucleic acid construct comprising a promotercapable of functioning in a plant cell operably linked to a nucleic acidsequence having a first nucleotide sequence comprising a fragment ofbetween about 100 nucleotides and about 400 nucleotides of a Nicotiananicotine demethylase polynucleotide and a second nucleotide sequencecapable of forming a double-stranded RNA with the first nucleotidesequence; and regenerating a transgenic Nicotiana plant. In someembodiments, the Nicotiana nicotine demethylase polynucleotide is atobacco nicotine demethylase polynucleotide.

The present invention also provides seed obtained from the transgenicNicotiana plant having a nicotine to nornicotine conversion rate of lessthan about 2%, where the seed comprises a heterologous nucleic acidconstruct comprising a promoter capable of functioning in a plant celloperably linked to a nucleic acid sequence having a first nucleotidesequence comprising a fragment of between about 100 nucleotides andabout 400 nucleotides of a Nicotiana nicotine demethylase polynucleotideand a second nucleotide sequence capable of forming a double-strandedRNA with the first nucleotide sequence, where the transgenic Nicotianaplants are transgenic converter lines of Nicotiana. In some embodiments,the Nicotiana nicotine demethylase polynucleotide is a tobacco nicotinedemethylase polynucleotide.

The present invention also provides transgenic plant cells comprising anucleic acid molecule having a promoter functional in a plant cell and anucleic acid sequence encoding a nicotine demethylase having anisoleucine residue at position 274 and a tryptophan residue at position330.

The present invention also provides methods of screening for a nicotinedemethylase sequence comprising: obtaining a nucleic acid sequence thathas greater than about 90% sequence identity with SEQ ID NO:5 andidentifying a codon sequence encoding for a tryptophan residue atposition 330 of the encoded polypeptide.

Further provided are methods for screening for a nicotine demethylasehaving an isoleucine at position 274 or a tryptophan at position 330comprising obtaining a nucleic acid sequence that has greater than about90% sequence identity with SEQ ID NO:5 and identifying a first codonsequence encoding for an isoleucine residue at position 274 or a secondcodon sequence encoding a tryptophan residue at position 330 of theencoded polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of nicotine, nornicotine, andN′-nitrosonornicotine (NNN).

FIG. 2 shows Northern blot analysis of converter and nonconverter RNAsusing 7D_A06 as a hybridization probe. Lanes 1 and 2 show RNAs isolatedfrom sodium bicarbonate-treated leaves of genotypes DH 98-325-5(nonconverter) and DH 98-325-6 (converter), respectively. Lanes 3 and 4show RNAs isolated from ethephon-treated leaves of genotypes DH 98-326-3(nonconverter) and DH 98-326-1 (converter), respectively. Estimated sizeof the hybridizing band is indicated in kilobases (kb).

FIG. 3A-3G shows a nucleotide sequence alignment of members of the3D_C12 gene family. Asterisks denote positions where sequence identityis conserved among all sequences compared. Positions where differencesare found are indicated with dashes and the corresponding residues areshaded grey. The nucleotide sequences present in the alignment include3D_C12 (SEQ ID NO:1), 3D_C12-10 (SEQ ID NO:3); 3D_C12-7 (SEQ ID NO:5);7D_A06 (SEQ ID NO:7); 3D_C12-15 (SEQ ID NO:9); and 131A_A02 (SEQ IDNO:11). The 3D_C12-15 and 131A_A02 entries are partial-length cDNAsequences. The 99 bp region of 3D_C12 that was used to make theRNAi-based construct is underlined.

FIG. 4 shows an alignment of predicted amino acid sequences forfull-length members of the 3D_C12 family of P450 genes. The amino acidsequences present in the alignment include 3D_C12 (SEQ ID NO:2),3D_C12-10 (SEQ ID NO:4); 3D_C12-7 (SEQ ID NO:6); and 7D_A06 (SEQ ID NO:8). Asterisks denote positions conserved among all four sequences.Residues that differ among the members are shaded in grey.

FIG. 5 shows a Northern blot analysis of transgenic plants possessingthe 3D_C12/RNAi construct. (A) Hybridization of the 3D_C12-7 probe toRNAs isolated from ethephon-treated, cured leaves of transgenic plantsdisplaying low nornicotine phenotypes (3D_C12/RNAi-1, 3, and 4) and highnornicotine phenotypes (3D_C12/RNAi-6, 7, and vector-only control plant11). Estimated size of hybridizing band is indicated in kilobases (kb).(B) Ethidium bromide staining of the portion of the gel used in (A) thatcontains the 28S ribosomal RNA to show the relative equivalence of RNAloading among the lanes.

FIG. 6 shows a Northern blot analysis of transgenic plants possessingsense-orientation constructs of members of the 3D_C12 gene family. (A)Hybridization of the 3D_C12-7 probe to RNAs isolated from nontreatedleaves of independent transgenic lines expressing 3D_C12-7, 3D_C12, and7D_A06 constructs and a vector-only control (control 8). Estimated sizeof hybridizing band is indicated in kilobases (kb). (B) Ethidium bromidestaining of the portion of the gel used in (A) that contains the 28Sribosomal RNA to show the relative equivalence of RNA loading among thelanes.

FIG. 7 shows a genomic sequence of a fragment of the 3D_C12-10 genepossessing an intron. Intron sequences are indicated in bold, italicizedtype. Exon sequences are shown in plain type. The sequencescorresponding to the PCR primers used to amplify the fragment from thetobacco genomic DNA are underlined.

FIG. 8 shows a diagram of the RNAi constructs used to silence expressionof of members of the 3D CD12 gene family.

DETAILED DESCRIPTION OF THE INVENTION Background and Definitions

Before describing the present invention in detail, it is to beunderstood that many modifications and other embodiments of theinventions set forth herein will come to mind to one skilled in the artto which this invention pertains having the benefit of the teachingspresented in the foregoing descriptions and the associated drawings.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Rather, these embodiments are provided so that thisdisclosure will satisfy applicable legal requirements.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Likenumbers refer to like elements throughout. Further, the article “a” and“an” are used herein to refer to one or more than one (i.e., to at leastone) of the grammatical object of the article. By way of example, “anelement” means one or more element. Throughout the specification theword “comprising,” or variations such as “comprises” or “comprising,”will be understood to imply the inclusion of a stated element, integeror step, or group of elements, integers or steps, but not the exclusionof any other element, integer or step, or group of elements, integers orsteps.

The present invention is drawn to compositions and methods forinhibiting the expression or function of cytochrome P450 polypeptidesthat are involved in the metabolic conversion of nicotine to nornicotinein a plant, particularly plants of the Nicotiana genus, includingtobacco plants of the various commercial varieties. As used herein, theterms “inhibit,” “inhibition,” and “inhibiting” are defined as anymethod known in the art or described herein that decreases theexpression or function of a gene product of interest (i.e., the targetgene product). “Inhibition” can be in the context of a comparisonbetween two plants, for example, a genetically altered plant versus awild-type plant. Alternatively, inhibition of expression or function ofa target gene product can be in the context of a comparison betweenplant cells, organelles, organs, tissues, or plant parts within the sameplant or between different plants, and includes comparisons betweendevelopmental or temporal stages within the same plant or plant part orbetween plants or plant parts. “Inhibition” includes any relativedecrement of function or production of a gene product of interest, up toand including complete elimination of function or production of thatgene product. The term “inhibition” encompasses any method orcomposition that down-regulates translation and/or transcription of thetarget gene product or functional activity of the target gene product.

The term “inhibitory sequence” encompasses any polynucleotide orpolypeptide sequence capable of inhibiting the expression or function ofa cytochrome P450 polypeptide involved in the metabolic conversion ofnicotine to nornicotine in a plant, such as full-length polynucleotideor polypeptide sequences, truncated polynucleotide or polypeptidesequences, fragments of polynucleotide or polypeptide sequences,variants of polynucleotide or polypeptide sequences, sense-orientednucleotide sequences, antisense-oriented nucleotide sequences, thecomplement of a sense- or antisense-oriented nucleotide sequence,inverted regions of nucleotide sequences, hairpins of nucleotidesequences, double-stranded nucleotide sequences, single-strandednucleotide sequences, combinations thereof, and the like. The term“polynucleotide sequence” includes sequences of RNA, DNA, chemicallymodified nucleic acids, nucleic acid analogs, combinations thereof, andthe like.

Inhibitory sequences are designated herein by the name of the targetgene product. Thus, a “cytochrome P450 inhibitory sequence” refers to aninhibitory sequence that is capable of inhibiting the expression of acytochrome P450 polypeptide that is involved in the metabolic conversionof nicotine to nornicotine in a plant, for example, at the level oftranscription and/or translation, or which is capable of inhibiting thefunction of such a cytochrome P450 polypeptide. When the phrase “capableof inhibiting” is used in the context of a polynucleotide inhibitorysequence, it is intended to mean that the inhibitory sequence itselfexerts the inhibitory effect; or, where the inhibitory sequence encodesan inhibitory nucleotide molecule (for example, hairpin RNA, miRNA, ordouble-stranded RNA polynucleotides), or encodes an inhibitorypolypeptide (i.e., a polypeptide that inhibits expression or function ofthe target gene product), following its transcription (for example, inthe case of an inhibitory sequence encoding a hairpin RNA, miRNA, ordouble-stranded RNA polynucleotide) or its transcription and translation(in the case of an inhibitory sequence encoding an inhibitorypolypeptide), the transcribed or translated product, respectively,exerts the inhibitory effect on the target gene product (i.e., inhibitsexpression or function of the target gene product).

By “host cell” is meant a cell that comprises a heterologous nucleicacid sequence of the invention. Though the nucleic acid sequences of theinvention, and fragments and variants thereof, can be introduced intoany cell of interest, of particular interest are plant cells, moreparticularly cells of a Nicotiana plant species, for example, thetobacco plant species and varieties described herein below.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

The term “variant” as used herein is intended to mean a substantiallysimilar sequence, and the term “native” polynucleotide or polypeptide isintended to mean a naturally occurring nucleotide sequence or amino acidsequence, respectively. By “fragment” is intended a portion of apolynucleotide or a portion of an amino acid sequence and hence proteinencoded thereby.

As used herein, the term “plant part” includes plant cells, plantprotoplasts, plant cell tissue cultures from which a whole plant can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants such as embryos, pollen, anthers, ovules,seeds, leaves, flowers, stems, branches, fruit, roots, root tips, andthe like. Progeny, variants, and mutants of regenerated plants are alsoincluded within the scope of the invention, provided that these comprisethe introduced nucleic acid sequences of the invention.

By “phenotypic change” is intended a measurable change in one or morecell functions. For example, plants having a genetic modification at thegenomic locus encoding a cytochrome P450 polypeptide of the inventioncan show reduced or eliminated expression or activity of that cytochromeP450 polypeptide.

The term “introducing” is intended to mean presenting to the plant thepolynucleotide or polypeptide in such a manner that the sequence gainsaccess to the interior of a cell of the plant.

The term “operably linked” is intended to mean a functional linkagebetween two or more elements. For example, an operable linkage between apolynucleotide of interest and a regulatory sequence (i.e., a promoter)is a functional link that allows for expression of the polynucleotide ofinterest. Operably linked elements may be contiguous or non-contiguous.When used to refer to the joining of two protein coding regions, byoperably linked is intended that the coding regions are in the samereading frame.

The term “heterologous” according to the present invention when used inreference to a sequence is intended to mean a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention. The term also is applicable to nucleicacid constructs, also referred to herein as “polynucleotide constructs”or “nucleotide constructs.” In this manner, a “heterologous” nucleicacid construct is intended to mean a construct that originates from aforeign species, or, if from the same species, is substantially modifiedfrom its native form in composition and/or genomic locus by deliberatehuman intervention. Heterologous nucleic acid constructs include, butare not limited to, recombinant nucleotide constructs that have beenintroduced into a plant or plant part thereof, for example, viatransformation methods or subsequent breeding of a transgenic plant withanother plant of interest.

For example, a promoter operably linked to a heterologous polynucleotideis from a species different from the species from which thepolynucleotide was derived, or, if from the same/analogous species, oneor both are substantially modified from their original form and/orgenomic locus, or the promoter is not the native promoter for theoperably linked polynucleotide. Furthermore, as used herein a chimericgene comprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

The term “expression” as used herein refers to the biosynthesis of agene product, including the transcription and/or translation of saidgene product. For example, for the purposes of the present invention, anexpression cassette, as described elsewhere herein, capable ofexpressing a polynucleotide that inhibits the expression of at least onecytochrome P450 polypeptide of the invention is an expression cassettecapable of producing an RNA molecule that inhibits the transcriptionand/or translation of at least one cytochrome P450 polypeptide. The“expression” or “production” of a protein or polypeptide from a DNAmolecule refers to the transcription and translation of the codingsequence to produce the protein or polypeptide, while the “expression”or “production” of a protein or polypeptide from an RNA molecule refersto the translation of the RNA coding sequence to produce the protein orpolypeptide.

Cytochrome 450 Polynucleotides and Polypeptides, and Variants andFragments Thereof

Compositions of the present invention include isolated cytochrome P450polynucleotides and polypeptides that are involved in the metabolicconversion of nicotine to nornicotine in plants, including commercialvarieties of tobacco plants. In particular, compositions of theinvention include isolated polypeptides comprising the amino acidsequences as shown in SEQ ID NOS:2, 4, 6, 8, 10, and 12, and isolatedpolynucleotides comprising the nucleotide sequences as shown in SEQ IDNOS:1, 3, 5, 7, 9, and 11. The polynucleotides of the invention find usein inhibiting expression of these cytochrome P450 polypeptides orvariants thereof that are involved in the metabolic conversion ofnicotine to nornicotine in plants, particularly tobacco plants.

In this manner, the invention further provides expression cassettescomprising all or a portion of the polynucleotide sequence set forth inSEQ ID NO:1, 3, 5, 7, 9, or 11, a complement or fragment thereof, or asequence having substantial sequence identity to SEQ ID NO:1, 3, 5, 7,9, or 11, or a complement or fragment thereof, operably linked to apromoter that is functional in a plant cell for use in expressing aninhibitory RNA transcript that interferes with expression (i.e.,transcription and/or translation) of cytochrome P450 polypeptidesdescribed herein. In some embodiments, the expression cassettes comprisethe nucleotide sequence as shown in SEQ ID NO:13, 14, 15, or 16, acomplement or fragment thereof, or a sequence having substantialsequence identity to SEQ ID NO:13, 14, 15, or 16, or a complement orfragment thereof. Introduction of these expression cassettes into aNicotiana plant of interest, particularly a tobacco plant of varietiescommonly known as flue or bright varieties, Burley varieties, darkvarieties, and oriental/Turkish varieties, results in the production oftobacco plants having reduced amounts of nornicotine and thenitrosamine, N′-nitrosonornicotine (NNN). Leaf and stem material fromthese transgenic plants can be used to produce a variety of tobaccoproducts having reduced levels of nornicotine, and a concomitantreduction in this carcinogenic nitrosamine metabolite.

The cytochrome P450 polynucleotides and encoded polypeptides of thepresent invention represent a novel cytochrome P450 gene family,designated the 3D_C12 cytochrome 450 gene family, that is newlyidentified as having a role in the metabolic conversion of nicotine tonornicotine in tobacco plants. Suppression of the expression of theirencoded gene products in transgenic tobacco plants results in asignificant reduction in the accumulation of nornicotine in the leavesof these transgenic plants. While not being bound by theory, themetabolic role of these polypeptides may be a direct one, i.e., directlycatalyzing the N-demethylation reaction, or an indirect one, i.e., inthe form of production of a product that leads to the up-regulation ofthe nicotine demethylase activity of the leaf. Regardless of themechanism, any means by which expression and/or function of thepolypeptides encoded by members of this cytochrome P450 gene family aretargeted for inhibition within a Nicotiana plant will be effective inreducing nornicotine levels, and levels of its carcinogenic metabolite,NNN, within leaves and stems of these plants.

The cytochrome P450 genes of the invention were isolated from tobaccolines of a Burley variety. The first of these cytochrome P450 genes,designated 3D_C12, encodes an mRNA transcript corresponding tonucleotides (nt) 1-1551 of the cDNA sequence set forth in SEQ ID NO:1,which codes for the full-length 517-residue polypeptide set forth in SEQID NO:2. The second member of this novel cytochrome P450 family,designated 3D_C12-10, encodes an mRNA transcript corresponding to nt1-1551 of the cDNA sequence set forth in SEQ ID NO:3, which codes forthe full-length 517-residue polypeptide set forth in SEQ ID NO:4. Thethird of these cytochrome P450 genes, designated 3D_C12-7, encodes anmRNA transcript corresponding to nt 1-1551 of the cDNA sequence setforth in SEQ ID NO:5, which codes for the full-length 517 residuepolypeptide set forth in SEQ ID NO:6. The fourth member of the novelcytochrome P450 family, designated 7D_A06, encodes an mRNA transcriptcorresponding to nt 1-1554 of the cDNA sequence set forth in SEQ IDNO:7, which codes for the full-length 518-residue polypeptide set forthin SEQ ID NO:8.

Two partial-length P450 gene sequences sharing high sequence identity tothe full-length members of the 3D_C12 cytochrome P450 gene family werealso isolated from these Burley tobacco lines. The first of these,designated 3D_C12-15, encodes an mRNA transcript corresponding to thecDNA sequence set forth in SEQ ID NO:9, which encodes the partial-lengthpolypeptide set forth in SEQ ID NO:10. The second partial-length P450gene sequence, designated 131A_A02, encodes an mRNA transcriptcorresponding to the cDNA sequence set forth in SEQ ID NO:11, whichencodes the partial-length polypeptide set forth in SEQ ID NO:12.

In one aspect, the cytochrome P450 genes of the present invention areinvolved in the conversion of nicotine to nornicotine in a plant. In oneaspect, the cytochrome P450 genes of the present invention have nicotinedemethylase activity.

An alignment of the members of the 3D_C12 cytochrome P450 gene family isshown in FIG. 3A-3G. The predicted amino acid sequences for the fourfull-length clones are aligned in FIG. 4. These sequences share highsequence identity with each other (at least 90% at both the nucleotideand amino acid level (see Tables 2 and 3, Example 4 herein below).

The invention encompasses isolated or substantially purifiedpolynucleotide or protein compositions. An “isolated” or “purified”polynucleotide or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide or protein as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide or protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Optimally, an “isolated” polynucleotide is freeof sequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, optimally culture medium represents less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Fragments of the disclosed cytochrome P450 polynucleotides andpolypeptides encoded thereby are also encompassed by the presentinvention. Fragments of a polynucleotide may encode protein fragmentsthat retain the biological activity of the native protein and hence areinvolved in the metabolic conversion of nicotine to nornicotine in aplant. Alternatively, fragments of a polynucleotide that are useful ashybridization probes or PCR primers using methods described belowgenerally do not encode fragment proteins retaining biological activity.Furthermore, fragments of the disclosed nucleotide sequences includethose that can be assembled within recombinant constructs for use ingene silencing with any method known in the art, including, but notlimited to, sense suppression/cosuppression, antisense suppression,double-stranded RNA (dsRNA) interference, hairpin RNA interference andintron-containing hairpin RNA interference, amplicon-mediatedinterference, ribozymes, and small interfering RNA or micro RNA, asdescribed herein below. Thus, fragments of a cytochrome P450polynucleotide sequence may range from at least about 20 nucleotides,about 50 nucleotides, about 70 nucleotides, about 100 nucleotides, about150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300nucleotides, about 350 nucleotides, about 400 nucleotides, and up to thefull-length polynucleotide encoding the proteins of the invention,depending upon the desired outcome. In one aspect, the fragments of acytochrome P450 polynucleotide sequence can be a fragment of betweenabout 50 and about 400 nucleotides, between about 70 and about 350nucleotides, between about 90 and about 325 nucleotides, between about90 and about 300 nucleotides, between about 90 and about 275nucleotides, between about 100 and about 400 nucleotides, between about100 and about 350 nucleotides, between about 100 and about 325nucleotides, between about 100 and about 300 nucleotides, between about125 and about 300 nucleotides, or between about 125 and about 275nucleotides in length. In some embodiments, a fragment of a cytochromeP450 polynucleotide is about 50, about 60, about 70, about 80, about 90,about 100, about 125, about 150, about 175, about 200, about 225, about250, about 275, about 300, about 325, about 350, about 400 nucleotidesin length, and other such values between about 70 and about 400nucleotides. In one such embodiment, a fragment of a cytochrome P450polynucleotide of the invention is about 90 bp to about 110 bp inlength, including 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, and 110 by in length. In another suchembodiment, a fragment of a cytochrome P450 polynucleotide of theinvention is about 290 to about 310 bp in length, including 290, 291,292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305,306, 307, 308, 309, and 310 by in length.

A fragment of a cytochrome P450 polynucleotide of the present inventionthat encodes a biologically active portion of a cytochrome P450polypeptide of the present invention will encode at least 15, 25, 30,50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500contiguous amino acids, or up to the total number of amino acids presentin a full-length cytochrome P450 polypeptide of the invention (e.g., 517for SEQ ID NOS: 2, 4, and 6; and 518 for SEQ ID NO:8), or will encode atleast 15, 25, 30, 50, 75, 100, 125, 150, or up to the total number ofamino acids present in a partial-length cytochrome P450 polypeptide ofthe invention (e.g., 173 for SEQ ID NO:10; and 222 for SEQ ID NO:12). Inone aspect, a fragment of a cytochrome P450 polynucleotide of theinvention encodes a polypeptide that comprises position 330 of theencoded polypeptide sequence. In another aspect, the polynucleotidefragment encodes a fragment of a cytochrome P450 polypeptide, where thepolypeptide fragment comprises the amino acids from position 225 throughthe amino acid at about position 600 of SEQ ID NO:6. In in one suchembodiment, the polynucleotide fragment encodes a fragment of acytochrome P450 polypeptide, where the polypeptide fragment comprisesthe amino acids from about position 239 through the amino acid at aboutposition 402 of SEQ ID NO:6. A biologically active portion of acytochrome P450 polypeptide can be prepared by isolating a portion ofone of the cytochrome P450 polynucleotides of the present invention,expressing the encoded portion of the cytochrome P450 polypeptide (e.g.,by recombinant expression in vitro), and assessing the activity of theencoded portion of the cytochrome P450 polypeptide, i.e., the ability topromote conversion of nicotine to nornicotine, using assays known in theart and those provided herein below.

Polynucleotides that are fragments of a cytochrome P450 nucleotidesequence of the present invention comprise at least 16, 20, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550, 1600, 1650, or 1700 contiguous nucleotides, or up to the number ofnucleotides present in a full-length cytochrome P450 polynucleotide asdisclosed herein (e.g., 539 for SEQ ID NO:9; 666 for SEQ ID NO:11; 1733for SEQ ID NOS:1, 3, and 5; and 1727 for SEQ ID NO:7). Polynucleotidesthat are fragments of a cytochrome P450 nucleotide sequence of thepresent invention comprise fragments from about 20 to about 1700contiguous nucleotides, from about 50 to about 1600 contiguousnucleotides, from about 75 to about 1500 contiguous nucleotides, fromabout 100 to about 1400 nucleotides, from about 150 to about 1300contiguous nucleotides, from about 150 to about 1200 contiguousnucleotides, from about 175 to about 1100 contiguous nucleotides, orfrom about 200 to about 1000 contiguous nucleotides from a cytochromeP450 polynucleotide as disclosed herein.

In one aspect, fragments of a cytochrome P450 polynucleotide comprise apolynucleotide sequence containing the nucleotides from about position700 to about position 1250 of a cytochrome P450 coding sequence. Inanother aspect, fragments of a cytochrome P450 polynucleotide comprise apolynucleotide sequence containing the nucleotides from about position715 to about position 1210, or from about position 717 to about position1207 of a cytochrome P450 coding sequence disclosed herein.

In other embodiments of the invention, fragments of a cytochrome P450polynucleotide comprise a polynucleotide sequence containing thenucleotides from about position 265 to about position 625 of acytochrome P450 coding sequence disclosed herein, or a complementthereof. In some of these embodiments, the fragments of a cytochromeP450 coding sequence disclosed herein comprise the nucleotidescorresponding to about position 297 to about position 594 of the P450coding sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, or a complementthereof.

In yet other embodiments of the invention, fragments of a cytochromeP450 polynucleotide comprise a polynucleotide sequence containing thenucleotides from about position 1420 to about position 1580 of acytochrome P450 coding sequence disclosed herein, or a complementthereof. In some of these embodiments, the fragments of a cytochromeP450 coding sequence disclosed herein comprise the nucleotidescorresponding to about position 1453 to about position 1551 of the P450coding sequence set forth in SEQ ID NO:1, or a complement thereof.

Variants of the disclosed polynucleotides and polypeptides encodedthereby are also encompassed by the present invention. Such naturallyoccurring variants include those variants that share substantialsequence identity to the disclosed cytochrome P450 polynucleotides andpolypeptides disclosed herein as defined herein below. The compositionsand methods of the invention can be used to target expression orfunction of any naturally occurring cytochrome P450 that sharessubstantial sequence identity to the disclosed cytochrome P450polypeptides and which possesses the relevant cytochrome P450 activity,i.e., involvement in the metabolic conversion of nicotine to nornicotinein plants. Such variants may result from, for example, geneticpolymorphism or from human manipulation as occurs with breeding andselection. Biologically active variants of a cytochrome P450 protein ofthe invention, for example, variants of the polypeptide set forth in SEQID NO:2, 4, 6, 8, 10, or 12, will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to the amino acid sequence forthe native protein as determined by sequence alignment programs andparameters described elsewhere herein, and are characterized by theirfunctional involvement in the metabolic conversion of nicotine tonornicotine in plants. A biologically active variant of a protein of theinvention may differ from that protein by as few as 1-15 amino acidresidues, as few as 10, as few as 9, as few as 8, as few as 7, as few as6, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1amino acid residue.

Variants of a particular polynucleotide of the present invention includethose naturally occurring polynucleotides that encode a cytochrome P450polypeptide that is involved in the metabolic conversion of nicotine tonornicotine in plants. Such polynucleotide variants can comprise adeletion and/or addition of one or more nucleotides at one or more siteswithin the native polynucleotide disclosed herein and/or a substitutionof one or more nucleotides at one or more sites in the nativepolynucleotide. Because of the degeneracy of the genetic code,conservative variants for polynucleotides include those sequences thatencode the amino acid sequence of one of the cytochrome P450polypeptides of the invention. Naturally occurring variants such asthese can be identified with the use of well-known molecular biologytechniques, as, for example, with polymerase chain reaction (PCR) andhybridization techniques as outlined below. Variant polynucleotides alsoinclude synthetically derived polynucleotides, such as those generated,for example, by using site-directed mutagenesis but which still sharesubstantial sequence identity to the naturally occurring sequencesdisclosed herein, and thus can be used in the methods of the inventionto inhibit the expression or function of a cytochrome P450 that isinvolved in the metabolic conversion of nicotine to nornicotine,including the cytochrome P450 polypeptides set forth in SEQ ID NOS:2, 4,6, and 8, and polypeptides comprising the sequence set forth in SEQ IDNO:10 or 12. Generally, variants of a particular polynucleotide of theinvention, for example, the sequence set forth in SEQ ID NO:1, 3, 5, 7,9, or 11, will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to that particular polynucleotide as determined bysequence alignment programs and parameters described elsewhere herein.

Variants of a particular polynucleotide of the present invention (alsoreferred to as the reference polynucleotide) can also be evaluated bycomparison of the percent sequence identity between the polypeptideencoded by the reference polynucleotide and the polypeptide encoded by avariant polynucleotide. For example, isolated polynucleotides encoding apolypeptide with a particular percent sequence identity to thefull-length polypeptide of SEQ ID NO:2, 4, 6, or 8, or thepartial-length polypeptide encoded by SEQ ID NO:9 or 11 are disclosed.Such polynucleotides can be used in the methods of the present inventionto target expression of cytochrome P450 polypeptides involved in themetabolic conversion of nicotine to nornicotine in plants, particularlytobacco plants, thereby inhibiting accumulation of nornicotine and itsmetabolite N′-nitrosonornicotine in the stems and leaves of thegenetically modified plant. Percent sequence identity between any twopolypeptides can be calculated using sequence alignment programs andparameters described elsewhere herein. Where any given pair ofpolynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In oneaspect, a variant polypeptide of the present invention includes apolypeptide having a tryptophan at position 330 or an isoleucine atposition 274 of the cytochrome P450 polypeptide, or both a tryptophan atposition 330 and an isoleucine at position 274.

Furthermore, the polynucleotides of the invention can be used to isolatecorresponding cytochrome P450 sequences from other organisms,particularly other plants, more particularly other members of theNicotiana genus. PCR, hybridization, and other like methods can be usedto identify such sequences based on their sequence homology to thesequences set forth herein. Sequences isolated based on their sequenceidentity to the nucleotide sequences set forth herein or to variants andfragments thereof are encompassed by the present invention. Suchsequences include sequences that are orthologs of the disclosedsequences.

According to the present invention, “orthologs” are genes derived from acommon ancestral gene and which are found in different species as aresult of speciation. Genes found in different species are consideredorthologs when their nucleotide sequences and/or their encoded proteinsequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions oforthologs are often highly conserved among species. Thus, isolatedpolynucleotides that encode for a cytochrome P450 polypeptide that isinvolved in the nicotine-to-nornicotine metabolic conversion and whichhybridize under stringent conditions to the cytochrome P450 sequencesdisclosed herein, or to variants or fragments thereof, are encompassedby the present invention. Such sequences can be used in the methods ofthe present invention to inhibit expression of cytochrome P450polypeptides that are involved in the metabolic conversion of nicotineto nornicotine in plants.

Using PCR, oligonucleotide primers can be designed for use in PCRreactions to amplify corresponding DNA sequences from cDNA or genomicDNA extracted from any plant of interest. Methods for designing PCRprimers and PCR cloning are generally known in the art and are disclosedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2ded., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). Innis etal., eds. (1990) PCR Protocols: A Guide to Methods and Applications(Academic Press, New York); Innis and Gelfand, eds. (1995) PCRStrategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially mismatchedprimers, and the like.

Hybridization techniques involve the use of all or part of a knownpolynucleotide as a probe that selectively hybridizes to othercorresponding polynucleotides present in a population of cloned genomicDNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism.

Hybridization may be carried out under stringent conditions. By“stringent conditions” or “stringent hybridization conditions” isintended conditions under which a probe will hybridize to its targetsequence to a detectably greater degree than to other sequences (e.g.,at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide.Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplaryhigh stringency conditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.Optionally, wash buffers may comprise about 0.1% to about 1% SDS.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours. The duration of the wash time will be atleast a length of time sufficient to reach equilibrium.

In a specific embodiment, stringency conditions include hybridization ina solution containing 5×SSC, 0.5% SDS, 5× Denhardt's, 0.45 μg/μl Poly ARNA, 0.45 μg/μl calf thymus DNA and 50% formamide at 42° C., and atleast one post-hybridization wash in a solution comprising from about0.01×SSC to about 1×SSC. The duration of hybridization is from about 14to about 16 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≥90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is optimal to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

Hybridization probes may be genomic DNA fragments, cDNA fragments, RNAfragments, or other oligonucleotides, and may be labeled with adetectable group such as ³²P, or any other detectable marker. Forexample, probes for hybridization can be made by labeling syntheticoligonucleotides based on the cytochrome P450 polynucleotides sequencesof the present invention. Methods for preparation of probes forhybridization and for construction of cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

For example, the cytochrome P450 polynucleotide sequences disclosedherein, or one or more portions thereof, may be used as probes capableof specifically hybridizing to corresponding cytochrome P450polynucleotides and messenger RNAs. To achieve specific hybridizationunder a variety of conditions, such probes include sequences that areunique among cytochrome P450 polynucleotide sequences, includingupstream regions 5′ to the coding sequence and downstream regions 3′ tothe coding sequence, and are optimally at least about 10 nucleotides inlength, and most optimally at least about 20 nucleotides in length. Suchprobes may be used to amplify corresponding cytochrome P450polynucleotides. This technique may be used to isolate additional codingsequences from a desired plant or as a diagnostic assay to determine thepresence of coding sequences in a plant. Hybridization techniquesinclude hybridization screening of plated DNA libraries (either plaquesor colonies; see, for example, Sambrook et al. (1989) Molecular Cloning:A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

As used herein, with respect to the sequence relationships between twoor more polynucleotides or polypeptides, the term “reference sequence”is a defined sequence used as a basis for sequence comparison. Areference sequence may be a subset or the entirety of a specifiedsequence; for example, as a segment of a full-length cDNA or genesequence, or the complete cDNA or gene sequence.

As used herein, the term “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two polynucleotides. Generally, the comparison window is at least20 contiguous nucleotides in length, and optionally can be 30, 40, 50,100, or longer. Those of skill in the art understand that to avoid ahigh similarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 arebased on the algorithm of Karlin and Altschul (1990) supra. BLASTnucleotide searches can be performed with the BLASTN program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleotidesequence encoding a protein of the invention. BLAST protein searches canbe performed with the BLASTX program, score=50, wordlength=3, to obtainamino acid sequences homologous to a protein or polypeptide of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST (in BLAST 2.0) can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST2.0) can be used to perform an iterated search that detects distantrelationships between molecules. See Altschul et al. (1997) supra. Whenutilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences, BLASTX forproteins) can be used (See www.ncbi.nlm.nih.gov). Alignment may also beperformed manually by inspection.

The sequence identity/similarity values provided herein were calculatedusing the BLASTX (Altschul et al. (1997) supra), Clustal W (Higgins etal. (1994) Nucleic Acids Res. 22: 4673-4680), and GAP (University ofWisconsin Genetic Computing Group software package) algorithms usingdefault parameters. The present invention also encompasses the use ofany equivalent program thereof for the analysis and comparison ofnucleic acid and protein sequences. By “equivalent program” is intendedany sequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by BLASTX, Clustal W, or GAP.

For purposes of the foregoing discussion of variant nucleotide andpolypeptide sequences encompassed by the present invention, the term“sequence identity” or “identity” in the context of two polynucleotidesor polypeptide sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window. When percentage of sequence identity isused in reference to proteins it is recognized that residue positionswhich are not identical often differ by conservative amino acidsubstitutions, where amino acid residues are substituted for other aminoacid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. When sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

The term “percentage of sequence identity” as used herein means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

When any two polypeptide sequences are optimally aligned for comparison,it is recognized that residues appearing opposite of one another withinthe alignment occupy positions within their respective polypeptides thatcorrespond to one another. Such positions are referred to herein as“corresponding positions” and the residues residing at correspondingpositions are referred to as “corresponding residues” or residues that“correspond” to one another. Thus, for example, where a polypeptide ofinterest is optimally aligned to a reference polypeptide sequencehaving, for example, 10 residues, the residue within the polypeptide ofinterest appearing opposite residue 5 of the reference sequence isreferred to as the “residue at the position corresponding to residue 5”of the reference sequence.

In like manner, when any two polynucleotide sequences are optimallyaligned for comparison, it is recognized that the nucleotides appearingopposite of one another within the alignment occupy positions withintheir respective polynucleotide positions that correspond to oneanother. Such positions are referred to herein as “correspondingpositions” and the nucleotides residing at corresponding positions arereferred to as “corresponding nucleotides” or nucleotides that“correspond” to one another. Thus, for example, where a polynucleotideof interest is optimally aligned to a reference polynucleotide sequencehaving, for example, 300 nucleotides, the nucleotide within thepolynucleotide of interest appearing opposite nucleotide 275 of thereference sequence is referred to as the “nucleotide at the positioncorresponding to nucleotide 275” of the reference sequence.

Where a region of nucleotides is being compared between a polynucleotideof interest and a reference polynucleotide, the nucleotides within theseregions are said to “correspond” to one another. Thus, for example,where a region of a reference polynucleotide sequence, for example, thepolynucleotide sequence set forth in SEQ ID NO:5, resides fromnucleotide position 265 to nucleotide position 625 of the referencepolynucleotide, and this region of nucleotides is being compared to thecorresponding region of nucleotides within an optimally alignedpolynucleotide sequence of interest, the nucleotides within thecorresponding region of the polynucleotide of interest are referred toherein as “a region” of the polynucleotide of interest that “correspondsto nucleotide position 265 to nucleotide position 625” of the referencesequence, in this case, SEQ ID NO:5.

Cytochrome P450 polynucleotide and polypeptide sequences can beidentified using the sequences provided herein. Such methods includeobtaining a polynucleotide or polypeptide sequence at least 80%, 85%,90%, 95%, 98%, 99% sequence identity with the polynucleotide sequence ofSEQ ID NO 1, 3, 5, 7, 9, or 11 or a complement or fragment thereof, or apolypeptide sequence of SEQ ID NO:2, 4, 6, 7, 10, or 12. In oneembodiment, the identified sequence contains or encodes for a tryptophanresidue at position 330 and an isoleucine residue at position 274.

In this manner, one aspect of the present invention is directed to amethod of screening for a nicotine demethylase sequence. This methodcomprises obtaining a nucleic acid sequence that has greater than about90% sequence identity with SEQ ID NO:3 or SEQ ID NO:5; and identifyingwithin this nucleic acid sequence a codon sequence encoding for atryptophan residue at position 330 of the polypeptide encoded by thenucleic acid sequence. In some embodiments, this nucleic acid sequencealso encodes an isoleucine at position 274 of the encoded polypeptide.Any suitable method known in the art can be used to identify the codonsequence encoding the tryptophan or isoleucine residue. In oneembodiment, the codon sequence is identified by a method selected fromthe group consisting of identifying a single nucleotide polymorphism andRT-PCR. In some embodiments of this aspect of the invention, thescreening method identifies a nicotine demethylase that convertsnicotine to nornicotine at a rate that is at least about 5-fold greaterthan the conversion rate of the nicotine demethylase encoded by SEQ IDNO:3 (polypeptide set forth in SEQ ID NO:4) or SEQ ID NO:5 (polypeptideset forth in SEQ ID NO:6). In particular embodiments, the nicotinedemethylase identified with this screening method has a conversion ratethat is at least about 8-fold greater than the conversion rate of thenicotine demethylase encoded by SEQ ID NO:3 or SEQ ID NO:5.

In another aspect, the present invention provides a method for screeningfor a nicotine demethylase having an isoleucine at position 274 or atryptophan at position 329 of the polypeptide. This method comprisesobtaining a nucleic acid sequence that has greater than about 90%sequence identity with SEQ ID NO:3 or SEQ ID NO:5; and identifying afirst codon sequence encoding for an isoleucine residue at position 274of the encoded polypeptide or a second codon sequence encoding atryptophan residue at position 330 of the encoded polypeptide. As notedabove, any suitable method known to those of skill in the art can beused to identify these codons. In one embodiment, either or both of thefirst and second codons are identified by a method selected from thegroup consisting of identifying a single nucleotide polymorphism andRT-PCR. In some embodiments of this aspect of the invention, thescreening method identifies a nicotine demethylase that convertsnicotine to nornicotine at a rate that is at least about 5-fold greaterthan the conversion rate of the nicotine demethylase encoded by SEQ IDNO:3 (polypeptide set forth in SEQ ID NO:4) or SEQ ID NO:5 (polypeptideset forth in SEQ ID NO:6). In particular embodiments, the nicotinedemethylase identified with this screening method has a conversion ratethat is at least about 8-fold greater than the conversion rate of thenicotine demethylase encoded by SEQ ID NO:3 or SEQ ID NO:5.

The present invention also provides transgenic plant cells, plants, andseed comprising a nucleic acid molecule having a promoter functional ina plant cell and a nucleic acid sequence encoding a nicotine demethylasehaving an isoleucine residue at position 274 and a tryptophan residue atposition 330. In some embodiments, the nucleic acid sequence encodingthis nicotine demethylase is derived from a sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7. Thesetransgenic plant cells, plants, and seed include, but are not limitedto, Nicotiana plant cells, Nicotiana plants, and seed of Nicotianaplants. In some embodiments, the Nicotiana plant cells, Nicotianaplants, and seed of Nicotiana plants are from converter Nicotianaplants.

Expression Cassettes For Use in the Methods of the Invention

Compositions of the present invention further include expressioncassettes comprising inhibitory sequences capable of inhibitingexpression or function of a cytochrome P450 polypeptide involved in theconversion of nicotine to nornicotine in a Nicotiana plant or plant partthereof, where the inhibitory sequences are operably linked to apromoter that is functional in a plant cell. In this manner, expressioncassettes comprising all or part of the sequence set forth in SEQ IDNO:1, 3, 5, 7, 9, or 11, a complement or fragment thereof, or sequencessharing substantial sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, ora complement or fragment thereof, operably linked to a promoter that isfunctional in a plant cell are constructed for use in the gene-silencingmethods of the present invention described herein below. Such sequencesare referred to herein as “inhibitory sequences” or “ inhibitorypolynucleotide sequences” as they are capable of being expressed as anRNA molecule that inhibits expression (i.e, transcription and/ortranslation) of the target cytochrome P450 polypeptide, for example, thepolypeptide set forth in SEQ ID NO:2, 4, 6, or 8 and variants thereof,or a polypeptide comprising the sequence set forth in SEQ ID NO:10 or 12and variants thereof, where the variant polypeptides have substantialsequence identity to these disclosed cytochrome P450 polypeptides andare involved in the metabolic conversion of nicotine to nornicotine in aplant.

As noted above, such inhibitory sequences include fragment sequences ofthe target cytochrome P450 polynucleotides. For example, a fragmentsequence can include any portion of the cytochrome P450 sequence,including coding and non-coding sequence (e.g., 5′ UTR, intron, and 3′UTR sequences), and can include fragments of between about 20 and about400 nucleotides, between about 50 and about 400 nucleotides, betweenabout 100 and about 400 nucleotides, between about 125 and about 325nucleotides, between about 125 and about 300 nucleotides, or betweenabout 125 and about 275 nucleotides.

In this manner, such inhibitory sequences include, but are not limited,sequences that comprise a fragment of a cytochrome P450 polynucleotidesequence ranging from at least about 20 nucleotides, about 50nucleotides, about 70 nucleotides, about 100 nucleotides, about 150nucleotides, about 200 nucleotides, about 250 nucleotides, about 300nucleotides, about 350 nucleotides, and up to the full-lengthpolynucleotide encoding the proteins of the invention, depending uponthe desired outcome. In one aspect, the inhibitory sequences comprise afragment of a cytochrome P450 polynucleotide sequence that is betweenabout 50 and about 400 nucleotides, between about 50 and about 350nucleotides, between about 70 and about 350 nucleotides, between about90 and about 325 nucleotides, between about 90 and about 300nucleotides, between about 90 and about 275 nucleotides, between about100 and about 400 nucleotides, between about 100 and about 350nucleotides, between about 100 and about 325 nucleotides, between about100 and about 300 nucleotides, between about 125 and about 300nucleotides, or between about 125 and about 275 nucleotides in length.In some embodiments, the inhibitory sequences comprise a fragment of acytochrome P450 polynucleotide sequence that is about 50, about 60,about 70, about 80, about 90, about 100, about 125, about 150, about175, about 200, about 225, about 250, about 275, about 300, about 325,about 350, about 400 nucleotides in length, and other such valuesbetween about 50 and about 400 nucleotides. It is recognized that theinhibitory sequence can also comprise a sequence that is complementaryto all or a part of the fragment of the cytochrome P450 polynucleotidesequence.

In one such embodiment, the inhibitory sequences comprise a fragment ofa cytochrome P450 polynucleotide of the invention that is about 90 bp toabout 110 bp in length, including 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110 bp inlength, and can also comprise a sequence that is complementary to all ora part of the fragment sequence. In another such embodiment, a fragmentof a cytochrome P450 polynucleotide of the invention is about 290 toabout 310 bp in length, including 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, and 310bp in length, and can also comprise a sequence that is complementary toall or a part of the fragment sequence.

In other embodiments of the invention, the inhibitory sequence within anexpression cassette of the invention comprises a polynucleotide sequencecontaining the nucleotides from about position 265 to about position 625of a cytochrome P450 coding sequence disclosed herein and sequence thatis fully or partially complementary thereto. In some of theseembodiments, the inhibitory sequence comprises the nucleotidescorresponding to about position 297 to about position 594 of the P450coding sequence set forth in SEQ ID NO:3 or SEQ ID NO:5 and a sequencethat is fully or partially complementary thereto. In preferredembodiments, such inhibitory sequences are expressed as a hairpin RNA asdescribed herein below.

In yet other embodiments of the invention, the inhibitory sequencewithin an expression cassette of the invention comprises apolynucleotide sequence containing the nucleotides from about position1420 to about position 1580 of a cytochrome P450 coding sequencedisclosed herein and a sequence that is fully or partially complementarythereto. In some of these embodiments, the inhibitory sequence comprisesthe nucleotides corresponding to about position 1453 to about position1551 of the P450 coding sequence set forth in SEQ ID NO:1 and a sequencethat is fully or partially complementary thereto. In preferredembodiments, such inhibitory sequences are expressed as a hairpin RNA asdescribed herein below.

It is recognized that expression cassettes of the present inventionencompass constructs in which a desired nucleic acid sequence isoperably linked to a promoter that is functional in a plant cell,particularly in the cell of a Nicotiana plant. By “promoter” is intendeda regulatory region of DNA usually comprising a TATA box capable ofdirecting RNA polymerase II to initiate RNA synthesis at the appropriatetranscription initiation site for a particular polynucleotide sequence.A promoter may additionally comprise other recognition sequencesgenerally positioned upstream or 5′ to the TATA box, referred to asupstream promoter elements, which influence the transcription initiationrate. It is also recognized that expression cassettes of the presentinvention encompass additional domains that modulate the level ofexpression, the developmental timing of expression, or tissue type thatexpression occurs in (e.g., Australian Patent No. AU-A-77751/94 and U.S.Pat. Nos. 5,466,785 and 5,635,618). By “functional” is intended thepromoter, when operably linked to an inhibitory sequence encoding aninhibitory nucleotide molecule (for example, a hairpin RNA,double-stranded RNA, miRNA polynucleotide, and the like), the promoteris capable of initiating transcription of the operably linked inhibitorysequence such that the inhibitory nucleotide molecule is expressed. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, or otherpromoters for expression in plants.

An expression cassette of the present invention may also contain atleast one additional gene to be cotransformed into the plant.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes. Such an expression cassette is provided with aplurality of restriction sites and/or recombination sites for insertionof the cytochrome P450 inhibitory polynucleotide sequence to be underthe transcriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

In this manner, an expression cassette of the present invention includesa transcriptional and translational initiation region (i.e., a promoter)in the 5′-3′ direction of transcription, an inhibitory sequence asdescribed elsewhere herein, and a transcriptional and translationaltermination region (i.e., termination region) functional in a plantcell. The regulatory regions (i.e., promoters, transcriptionalregulatory regions, and translational termination regions) and/or theinhibitory sequence of the invention may be native/analogous to the hostcell or to each other. Alternatively, the regulatory regions and/or theinhibitory sequence of the invention may be heterologous to the hostcell or to each other. While heterologous promoters can be used toexpress the inhibitory sequences of the invention, native promotersequences may also be used.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked inhibitory sequence ofthe invention, may be native with the plant host, or may be derived fromanother source (i.e., foreign or heterologous to the promoter, theinhibitory sequence of interest, the plant host, or any combinationthereof). Convenient termination regions are available from theTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase termination regions. See also Guerineau et al. (1991) Mol. Gen.Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al.(1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272;Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic AcidsRes. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res.15:9627-9639.

Expression cassettes of the present invention may additionally contain5′ leader sequences that can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus) (Virology 154:9-20), and human immunoglobulin heavy-chain bindingprotein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4)(Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader(TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV)(Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa etal. (1987) Plant Physiol. 84:965-968. Other methods known to enhancetranslation can also be utilized.

In preparing the expression cassette, DNA fragments of the invention maybe manipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Adaptersor linkers may be employed to join the DNA fragments or othermanipulations may be involved to provide for convenient restrictionsites, removal of superfluous DNA, removal of restriction sites, or thelike. For this purpose, in vitro mutagenesis, primer repair,restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

The expression cassettes of the present invention can also comprise aselectable marker gene for the selection of transformed cells.Selectable marker genes are utilized for the selection of transformedcells or tissues. Marker genes include genes encoding antibioticresistance, such as those encoding neomycin phosphotransferase II (NEO)and hygromycin phosphotransferase (HPT), as well as genes conferringresistance to herbicidal compounds, such as glufosinate ammonium,bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).Additional selectable markers include phenotypic markers such asβ-galactosidase and fluorescent proteins such as green fluorescentprotein (GFP) (Rouwendal et al. (1997) Plant Mo. Biol. 33:989-999; Su etal. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) PlantCell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J.Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol129:913-42), and yellow fluorescent protein (PhiYFP™ from Evrogen, see,Bolte et al. (2004) J. Cell Science 117:943-954). For additionalselectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech.3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol.Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp.177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724. Such disclosures are herein incorporatedby reference. The above list of selectable marker genes is not meant tobe limiting. Any selectable marker gene can be used in the presentinvention.

A number of promoters can be used in the practice of the invention. Ofparticular interest are constitutive promoters, inducible promoters,particularly chemical-inducible promoters, and tissue-preferredpromoters, particularly leaf-preferred promoters.

Chemical-inducible promoters can be used to inhibit the expression of acytochrome P450 that is involved in the metabolic conversion of nicotineto nornicotine in a plant through the application of an exogenouschemical regulator. Chemical-inducible promoters are known in the artand include, but are not limited to, the tobacco PR-1a promoter, whichis activated by salicylic acid. Other chemical-inducible promoters ofinterest include steroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl.Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J.14(2):247-257) and tetracycline-inducible promoters (see, for example,Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos.5,814,618 and 5,789,156), herein incorporated by reference.

Constitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in U.S. Pat.No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature313:810-812); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, U.S.Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to target expression of aninhibitory polynucleotide sequence of the present invention within aparticular plant tissue. Tissue-preferred promoters include Yamamoto etal. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant CellPhysiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet.254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al.(1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) PlantPhysiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505.

Of particular interest are leaf-preferred promoters that provide forexpression predominately in leaf tissues. See, for example, Yamamoto etal. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol.105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778;Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; Baszczynski et al. (1988) Nucl. Acid Res.16:4732; Mitra et al. (1994) Plant Molecular Biology 26:35-93; Kayaya etal. (1995) Molecular and General Genetics 248:668-674; and Matsuoka etal. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.Senecence-regulated promoters are also of use, such as SAM22 (Crowell etal. (1992) Plant Mol. Biol. 18:459-466); SAG12 (Lohman et al. (1994)Physiol. Plant. 92:322-328; Wingler et al. (1998) Plant Physiol.116:329-335); SAG 13 (Gan and Amasino (1997) Plant Physiol. 113:313-319;SAG15 (Gan (1995) “Molecular Characterization and Genetic Manipulationof Plant Senescence,” Ph.D. Thesis, University of Wisconsin, Madison);SEN1 (Oh et al. (1996) Plant Mol. Biol. 30:739-754; promoter of asenescence-specific gene for expression of IPT (Gan and Amasino 91995)Science 270:1986-1988); and the like (see, for example, Or: et al.(1999) Plant Cell 11:1073-1080 and McCabe et al. (2001) Plant Physiol.127:505-516).

Methods For Inhibiting Expression or Function of a Cytochrome P450Involved in the Conversion of Nicotine to Nornicotine

Methods of reducing the concentration, content, and/or activity of acytochrome P450 polypeptide of the present invention in a Nicotianaplant or plant part, particularly the leaf tissue, are provided. Manymethods may be used, alone or in combination, to reduce or eliminate theactivity of a cytochrome P450 polypeptide of the present invention. Inaddition, combinations of methods may be employed to reduce or eliminatethe activity of two or more different cytochrome P450 polypeptides.

In accordance with the present invention, the expression of a cytochromeP450 polypeptide of the present invention is inhibited if the proteinlevel of the cytochrome P450 polypeptide is statistically lower than theprotein level of the same cytochrome P450 polypeptide in a plant thathas not been genetically modified or mutagenized to inhibit theexpression of that cytochrome P450 polypeptide, and where these plantshave been cultured and harvested using the same protocols. In particularembodiments of the invention, the protein level of the cytochrome P450polypeptide in a modified plant according to the invention is less than95%, less than 90%, less than 80%, less than 70%, less than 60%, lessthan 50%, less than 40%, less than 30%, less than 20%, less than 10%,less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%of the protein level of the same cytochrome P450 polypeptide in a plantthat is not a mutant or that has not been genetically modified toinhibit the expression of that cytochrome P450 polypeptide and which hasbeen cultured and harvested using the same protocols. The expressionlevel of the cytochrome P450 polypeptide may be measured directly, forexample, by assaying for the level of the cytochrome P450 transcript orcytochrome P450 polypeptide expressed in the Nicotiana plant or plantpart, or indirectly, for example, by measuring the conversion ofnicotine to nornicotine in the Nicotiana plant or plant part. Methodsfor monitoring the expression level of a protein are known in the art,and include, but are not limited to, Northern blot analysis as discussedin the examples herein below. Methods for determining the activity ofthe targeted cytochrome P450 polypeptide in converting nicotine tonornicotine are described elsewhere herein below, and include, but arenot limited to, alkaloid analysis using gas chromatography, for examplethe procedures described in the examples herein below.

In other embodiments of the invention, the activity of one or morecytochrome P450 polypeptides is reduced or eliminated by transforming aplant or plant part with an expression cassette comprising apolynucleotide encoding a polypeptide that inhibits the activity of oneor more cytochrome P450 polypeptides of the present invention. Theactivity of a cytochrome P450 polypeptide in converting nicotine tonornicotine in a Nicotiana plant or plant part is inhibited according tothe present invention if this conversion activity is statistically lowerthan conversion activity of the same cytochrome P450 polypeptide in aNicotiana plant or plant part that has not been genetically modified toinhibit the conversion activity of that cytochrome P450 polypeptide andwhich has been cultured and harvested using the same protocols. Inparticular embodiments, activity of a cytochrome P450 polypeptide inconverting nicotine to nornicotine in a modified Nicotiana plant orplant part according to the invention is inhibited if the activity isless than 95%, less than 90%, less than 80%, less than 70%, less than60%, less than 50%, less than 40%, less than 30%, less than 20%, lessthan 10%, less than 5%, less than 4%, less than 3%, less than 2%, orless than 1% of the conversion activity of the same cytochrome P450polypeptide in a Nicotiana plant that that has not been geneticallymodified to inhibit the expression of that cytochrome P450 polypeptideand which has been cultured and harvested using the same protocols. Theactivity of a cytochrome P450 polypeptide in converting nicotine tonornicotine in a Nicotiana plant or plant part is eliminated accordingto the invention when it is not detectable by the assay methodsdescribed elsewhere herein. Methods of determining the activity of acytochrome P450 polypeptide in converting nicotine to nornicotine in aNicotiana plant or plant part are described elsewhere herein, andinclude the alkaloid analyses using gas chromatography disclosed in theexamples herein below.

In specific embodiments, a cytochrome P450 inhibitory polynucleotidesequence described herein is introduced into a Nicotiana plant or plantpart. Subsequently, a Nicotiana plant or plant part having theintroduced inhibitory polynucleotide sequence of the invention isselected using methods known to those of skill in the art such as, butnot limited to, Southern blot analysis, DNA sequencing, PCR analysis, orphenotypic analysis. A plant or plant part altered or modified by theforegoing embodiments is grown under plant forming conditions for a timesufficient to modulate the concentration and/or activity of polypeptidesof the present invention in the plant. Plant forming conditions are wellknown in the art and discussed briefly elsewhere herein.

In some embodiments, a transformed tobacco plant containing a cytochromeP450 inhibitory polynucleotide sequence described herein has a reducedlevel of conversion of nicotine to nornicotine. In particularembodiments, conversion of nicotine to nornicotine in a transformedtobacco plant or plant part according to the invention is less than 95%,less than 90%, less than 80% less than 70%, less than 60%, less than50%, less than 40%, less than 30%, less than 20% less than 10%, lessthan 5%, less than 4%, less than 3%, less than 2%, or less than 1% ofthe conversion in a tobacco plant that that has not been geneticallymodified to inhibit the expression of that cytochrome P450 polypeptideand which has been cultured and harvested using the same protocols. Insome embodiments, the transformed tobacco plant is a converter tobaccoplant. In some embodiments, the transformed tobacco plant has aconversion rate lower than the rate observed in commercial nonconvertertobacco plants.

It is also recognized that the level and/or activity of the polypeptidemay be modulated by employing a polynucleotide that is not capable ofdirecting, in a transformed plant, the expression of a protein or anRNA. For example, the polynucleotides of the invention may be used todesign polynucleotide constructs that can be employed in methods foraltering or mutating a genomic nucleotide sequence in an organism. Suchpolynucleotide constructs include, but are not limited to, RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides, and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use are known in the art. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984;all of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc.Natl. Acad. Sci. USA 96:8774-8778; herein incorporated by reference.

It is therefore recognized that methods of the present invention do notdepend on the incorporation of the entire cytochrome P450 inhibitorypolynucleotide into the genome, only that the Nicotiana plant or plantpart thereof is altered as a result of the introduction of thisinhibitory polynucleotide into a cell. In one embodiment of theinvention, the genome may be altered following the introduction of thecytochrome P450 inhibitory polynucleotide into a cell. For example, theinhibitory polynucleotide, or any part thereof, may incorporate into thegenome of the plant. Alterations to the genome include, but are notlimited to, additions, deletions, and substitutions of nucleotides intothe genome. While the methods of the present invention do not depend onadditions, deletions, and substitutions of any particular number ofnucleotides, it is recognized that such additions, deletions, orsubstitutions comprise at least one nucleotide.

It is further recognized that reducing the level and/or activity of acytochrome P450 sequence of the present invention can be performed toelicit the effects of the sequence only during certain developmentalstages and to switch the effect off in other stages where expression isno longer desirable. Control of cytochrome P450 expression can beobtained via the use of inducible or tissue-preferred promoters.Alternatively, the gene could be inverted or deleted using site-specificrecombinases, transposons or recombination systems, which would alsoturn on or off expression of the cytochrome P450 sequence.

According to the present invention, changes in levels, ratios, activity,or distribution of cytochrome P450 polypeptides of the presentinvention, or changes in Nicotiana plant or plant part phenotype,particularly reduced accumulation of nornicotine and its carcinogenicmetabolite, NNN, could be measured by comparing a subject plant or plantpart to a control plant or plant part, where the subject plant or plantpart and the control plant or plant part have been cultured and/orharvested using the same protocols. As used herein, a subject plant orplant part is one in which genetic alteration, such as transformation,has been affected as to the cytochrome P450 polypeptide of interest, oris a Nicotiana plant or plant part that is descended from a Nicotianaplant or plant part so altered and which comprises the alteration. Acontrol plant or plant part provides a reference point for measuringchanges in phenotype of the subject plant or plant part.

The measurement of changes in phenotype can be measured at any time in aplant or plant part, including during plant development, senescence, orafter curing. In other embodiments, the measurement of changes inphenotype can be measured in plants grown under any conditions,including from plants grown in a growth chamber, greenhouse, or in afield. In one embodiment, changes in phenotype can be measured bydetermining the nicotine to nornicotine conversion rate. In a preferredembodiment, conversion can be measured by dividing the percentage ofnornicotine (as a percentage of the total tissue weight) by the sum ofthe percentage nicotine and nornicotine (as percentages of the totaltissue weight) and multiplying by 100.

According to the present invention, a control plant or plant part maycomprise a wild-type Nicotiana plant or plant part, i.e., of the samegenotype as the starting material for the genetic alteration thatresulted in the subject plant or plant part. A control plant or plantpart may also comprise a Nicotiana plant or plant part of the samegenotype as the starting material but that has been transformed with anull construct (i.e., with a construct that has no known effect on thetrait of interest, such as a construct comprising a selectable markergene). Alternatively, a control plant or plant part may comprise aNicotiana plant or plant part that is a non-transformed segregant amongprogeny of a subject plant or plant part, or a Nicotiana plant or plantpart genetically identical to the subject plant or plant part but thatis not exposed to conditions or stimuli that would induce suppression ofthe cytochrome P450 gene of interest. Finally, a control plant or plantpart may comprise the subject plant or plant part itself underconditions in which the cytochrome P450 inhibitory sequence is notexpressed. In all such cases, the subject plant or plant part and thecontrol plant or plant part are cultured and harvested using the sameprotocols.

As described elsewhere herein, methods are provided to reduce oreliminate the activity and/or concentration of a cytochrome P450polypeptide of the present invention by introducing into a Nicotianaplant or plant part a cytochrome P450 inhibitory polynucleotide sequencethat is capable of inhibiting expression or function of a cytochromeP450 polypeptide that is involved in the metabolic conversion ofnicotine to nornicotine. In some embodiments, the inhibitory sequence isintroduced by transformation of the plant or plant part, such as a plantcell, with an expression cassette that expresses a polynucleotide thatinhibits the expression of the cytochrome P450 polypeptide. Thepolynucleotide may inhibit the expression of a cytochrome P450polypeptide directly, by preventing translation of the cytochrome P450polypeptide messenger RNA, or indirectly, by encoding a polypeptide thatinhibits the transcription or translation of an cytochrome P450polypeptide gene encoding a cytochrome P450 polypeptide. Methods forinhibiting or eliminating the expression of a gene product in a plantare well known in the art, and any such method may be used in thepresent invention to inhibit the expression of cytochrome P450polypeptides.

In other embodiments, the activity of a cytochrome P450 polypeptide ofthe present invention may be reduced or eliminated by disrupting thegene encoding the cytochrome P450 polypeptide. The invention encompassesmutagenized plants that carry mutations in cytochrome P450 genes, wherethe mutations reduce expression of the cytochrome P450 gene or inhibitthe activity of an encoded cytochrome P450 polypeptide of the presentinvention.

In some embodiments of the present invention, a Nicotiana plant or plantpart is transformed with an expression cassette that is capable ofexpressing a polynucleotide that inhibits the expression of a cytochromeP450 sequence. Such methods may include the use of sensesuppression/cosuppression, antisense suppression, double-stranded RNA(dsRNA) interference, hairpin RNA interference and intron-containinghairpin RNA interference, amplicon-mediated interference, ribozymes, andsmall interfering RNA or micro RNA.

For cosuppression, an expression cassette is designed to express an RNAmolecule corresponding to all or part of a messenger RNA encoding acytochrome P450 polypeptide of interest (for example, a cytochrome P450polypeptide comprising the sequence set forth in SEQ ID NO:2, 4, 6, 8,10, or 12 or a sequence having substantial sequence identity to SEQ IDNO:2, 4, 6, 8, 10, or 12) in the “sense” orientation. Over expression ofthe RNA molecule can result in reduced expression of the native gene.Multiple plant lines transformed with the cosuppression expressioncassette are then screened to identify those that show the greatestinhibition of cytochrome P450 polypeptide expression.

The polynucleotide used for cosuppression may correspond to all or partof the sequence encoding a cytochrome P450 polypeptide or the presentinvention, all or part of the 5′ and/or 3′ untranslated region of acytochrome P450 polypeptide transcript, or all or part of both thecoding sequence and the untranslated regions of a transcript encoding acytochrome P450 polypeptide. In some embodiments where thepolynucleotide comprises all or part of the coding region for acytochrome P450 polypeptide of the present invention, the expressioncassette is designed to eliminate the start codon of the polynucleotideso that no protein product will be transcribed.

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable protein levels for the proteinsencoded by these genes or may also be used to inhibit the expression ofmultiple proteins in the same plant (e.g., Broin et al. (2002) PlantCell 14:1417-1432; U.S. Pat. No. 5,942,657). Methods for usingcosuppression to inhibit the expression of endogenous genes in plantsare described in Flavell et al. (1994) Proc. Natl. Acad. Sci. USA91:3490-3496; Jorgensen et al. (1996) Plant Mol. Biol. 31:957-973;Johansen and Carrington (2001) Plant Physiol. 126:930-938; Broin et al.(2002) Plant Cell 14:1417-1432; Stoutjesdijk et al (2002) Plant Physiol.129:1723-1731; Yu et al. (2003) Phytochemistry 63:753-763; and U.S. Pat.Nos. 5,034,323, 5,283,184, and 5,942,657; each of which is hereinincorporated by reference. The efficiency of cosuppression may beincreased by including a poly-dT region in the expression cassette at aposition 3′ to the sense sequence and 5′ of the polyadenylation signal.See, U.S. Patent Publication No. 20020048814, herein incorporated byreference. Typically, such a nucleotide sequence has substantialsequence identity to the sequence of the transcript of the endogenousgene, optimally greater than about 65% sequence identity, more optimallygreater than about 85% sequence identity, most optimally greater thanabout 95% sequence identity (e.g., U.S. Pat. Nos. 5,283,184 and5,034,323; herein incorporated by reference).

In some embodiments of the invention, inhibition of the expression ofthe cytochrome P450 polypeptide of the present invention may be obtainedby antisense suppression. For antisense suppression, the expressioncassette is designed to express an RNA molecule complementary to all orpart of a messenger RNA encoding the cytochrome P450 polypeptide. Overexpression of the antisense RNA molecule can result in reducedexpression of the native gene. Accordingly, multiple plant linestransformed with the antisense suppression expression cassette arescreened to identify those that show the greatest inhibition ofcytochrome P450 polypeptide expression.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the cytochromeP450 polypeptide, all or part of the complement of the 5′ and/or 3′untranslated region of the cytochrome P450 polypeptide transcript, orall or part of the complement of both the coding sequence and theuntranslated regions of a transcript encoding the cytochrome P450polypeptide. In addition, the antisense polynucleotide may be fullycomplementary (i.e., 100% identical to the complement of the targetsequence) or partially complementary (i.e., less than 100% identical tothe complement of the target sequence) to the target sequence. Antisensesuppression may be used to inhibit the expression of multiple proteinsin the same plant (e.g., U.S. Pat. No. 5,942,657). Furthermore, portionsof the antisense nucleotides may be used to disrupt the expression ofthe target gene. Generally, sequences of at least 50 nucleotides, 100nucleotides, 200 nucleotides, 300, 400, 450, 500, 550, or greater may beused. Methods for using antisense suppression to inhibit the expressionof endogenous genes in plants are described, for example, in Liu et al(2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and5,942,657, each of which is herein incorporated by reference. Efficiencyof antisense suppression may be increased by including a poly-dT regionin the expression cassette at a position 3′ to the antisense sequenceand 5′ of the polyadenylation signal. See, U.S. Patent Publication No.20020048814, herein incorporated by reference.

For dsRNA interference, a sense RNA molecule like that described abovefor cosuppression and an antisense RNA molecule that is fully orpartially complementary to the sense RNA molecule are expressed in thesame cell, resulting in inhibition of the expression of thecorresponding endogenous messenger RNA.

Expression of the sense and antisense molecules can be accomplished bydesigning the expression cassette to comprise both a sense sequence andan antisense sequence for the target cytochrome P450 sequence.Alternatively, separate expression cassettes may be used for the senseand antisense sequences. Multiple plant lines transformed with the dsRNAinterference expression cassette or expression cassettes are thenscreened to identify plant lines that show the greatest inhibition ofexpression of the targeted cytochrome P450 polypeptide. Methods forusing dsRNA interference to inhibit the expression of endogenous plantgenes are described in Waterhouse et al. (1998) Proc. Natl. Acad. Sci.USA 95:13959-13964, Liu et al. (2002) Plant Physiol. 129:1732-1743, andWO 99/49029, WO 99/53050, WO 99/61631, and WO 00/49035; each of which isherein incorporated by reference.

Amplicon expression cassettes comprise a plant virus-derived sequencethat contains all or part of the target gene but generally not all ofthe genes of the native virus. The viral sequences present in thetranscription product of the expression cassette allow the transcriptionproduct to direct its own replication. The transcripts produced by theamplicon may be either sense or antisense relative to the targetsequence (i.e., the messenger RNA for a cytochrome P450 polypeptide thatis involved in the metabolic conversion of nicotine to nornicotine).Methods of using amplicons to inhibit the expression of endogenous plantgenes are described, for example, in Angell and Baulcombe (1997) EMBO J.16:3675-3684, Angell and Baulcombe (1999) Plant J. 20:357-362, and U.S.Pat. No. 6,646,805, each of which is herein incorporated by reference.

In additional embodiments of the present invention, the polynucleotideexpressed by the expression cassette of the invention is catalytic RNAor has ribozyme activity specific for the messenger RNA of a cytochromeP450 polypeptide described herein. Thus, the polynucleotide causes thedegradation of the endogenous messenger RNA, resulting in reducedexpression of the cytochrome P450 polypeptide. This method is described,for example, in U.S. Pat. No. 4,987,071, herein incorporated byreference.

In further embodiments of the invention, inhibition of the expression ofone or more cytochrome P450 polypeptides may be obtained by RNAinterference (RNAi) by expression of a gene encoding a micro RNA(miRNA). miRNAs are regulatory agents consisting of about 22ribonucleotides. miRNA are highly efficient at inhibiting the expressionof endogenous genes. See, for example Javier et al. (2003) Nature 425:257-263, herein incorporated by reference.

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous miRNA gene. The miRNAgene encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to another endogenous gene(target sequence). For suppression of cytochrome P450 polypeptideexpression, the 22-nucleotide sequence is selected from a cytochromeP450 polypeptide transcript sequence and contains 22 nucleotidesencoding this cytochrome P450 polypeptide sequence in sense orientationand 21 nucleotides of a corresponding antisense sequence that iscomplementary to the sense sequence. miRNA molecules are highlyefficient at inhibiting the expression of endogenous genes, and the RNAinterference they induce is inherited by subsequent generations ofplants.

In still other embodiments of the invention, inhibition of theexpression of one or more cytochrome P450 polypeptides by RNAi may beobtained by hairpin RNA (hpRNA) interference or intron-containinghairpin RNA (ihpRNA) interference. These methods are highly efficient atinhibiting the expression of endogenous genes. See, Waterhouse andHelliwell (2003) Nat. Rev. Genet. 4:29-38 and the references citedtherein.

For hpRNA interference, the expression cassette is designed to expressan RNA molecule that hybridizes with itself to form a hairpin structurethat comprises a single-stranded loop region and a base-paired stem. Thebase-paired stem region comprises a sense sequence corresponding to allor part of the endogenous messenger RNA encoding the gene product whoseexpression is to be inhibited, in this case, a cytochrome P450polypeptide described herein, and an antisense sequence that is fully orpartially complementary to the sense sequence. Alternatively, thebase-paired stem region may correspond to a portion of a promotersequence controlling expression of the gene encoding the cytochrome P450polypeptide to be inhibited. Thus, the base-paired stem region of themolecule generally determines the specificity of the RNA interference.hpRNA molecules are highly efficient at inhibiting the expression ofendogenous genes, and the RNA interference they induce is inherited bysubsequent generations of plants. See, for example, Chuang andMeyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijket al. (2002) Plant Physiol. 129:1723-1731; and Waterhouse and Helliwell(2003) Nat. Rev. Genet. 4:29-38. Methods for using hpRNA interference toinhibit or silence the expression of genes are described, for example,in Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;Stoutjesdijk et al. (2002) Plant Physiol. 129:1723-1731; Waterhouse andHelliwell (2003) Nat. Rev. Genet. 4:29-38; Pandolfini et al. BMCBiotechnology 3:7, and U.S. Patent Publication No. 20030175965; each ofwhich is herein incorporated by reference. A transient assay for theefficiency of hpRNA constructs to silence gene expression in vivo hasbeen described by Panstruga et al. (2003) Mol. Biol. Rep. 30:135-140,herein incorporated by reference.

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA, but the RNA molecule additionally comprises an intron that iscapable of being spliced in the cell in which the ihpRNA is expressed.The use of an intron minimizes the size of the loop in the hairpin RNAmolecule following splicing, and this increases the efficiency ofinterference. See, for example, Smith et al. (2000) Nature 407:319-320.In fact, Smith et al. show 100% suppression of endogenous geneexpression using ihpRNA-mediated interference. Methods for using ihpRNAinterference to inhibit the expression of endogenous plant genes aredescribed, for example, in Smith et al. (2000) Nature 407:319-320;Wesley et al. (2001) Plant J. 27:581-590; Wang and Waterhouse (2001)Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell (2003) Nat.Rev. Genet. 4:29-38; Helliwell and Waterhouse (2003) Methods 30:289-295,and U.S. Pat. Publication No. 20030180945, each of which is hereinincorporated by reference.

In one such embodiment, RNAi is accomplished by expressing an inhibitorysequence that comprises a first sequence of a cytochrome P450polynucleotide of the invention that is about 90 bp to about 110 bp inlength, including 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, and 110 by in length, and a secondsequence that is complementary to all or a part of the first sequence.In another such embodiment, the inhibitory sequence comprises a firstsequence of a cytochrome P450 polynucleotide of the invention that isabout 290 to about 310 bp in length, including 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, and 310 bp in length, and a second sequence that is complementaryto all or a part of the fragment sequence.

In other embodiments of the invention, RNAi is accomplished byexpressing an inhibitory sequence that comprises a first polynucleotidesequence containing the nucleotides from about position 265 to aboutposition 625 of a cytochrome P450 coding sequence disclosed herein and asecond sequence that is fully or partially complementary thereto. Insome of these embodiments, the inhibitory sequence comprises as thefirst polynucleotide sequence the nucleotides corresponding to aboutposition 297 to about position 594 of the P450 coding sequence set forthin SEQ ID NO:3 or SEQ ID NO:5 and the second sequence is the complement(i.e., antisense sequence) of this first sequence. The inhibitorysequence can optionally comprise an intron sequence linked between thefirst and second sequences. Any intron known to those of skill in theart can be used in this manner. In some embodiments, the intron is fromthe soybean omega-6 fatty acid desaturase (FAD) (see GenBank AccessionNo. DQ672337, and Example 7 herein below). In one such embodiment, theintron comprises about 151 nucleotides that comprise nucleotides 100-247of the soybean omega-6 fatty acid desaturase polynucleotide shown inGenBankk Accession No. DQ672337. Examples of other introns include, butare not limited to, the intron nucleotide sequences of alcoholdehydrogenase (adh1) genes. Expression of this inhibitory sequenceproduces an intron-containing hairpin RNA that strongly interferes withexpression of the cytochrome P450 polypeptides disclosed herein. In thismanner, Nicotiana plants that are normally converters of nicotine tonornicotine that are transformed with an expression cassette comprisingsuch an inhibitory sequence advantageously have a nicotine tonornicotine conversion rate that, surprisingly, is even lower than thatobserved for Nicotiana plants that are nonconverters of nicotine tonornicotine.

In yet other embodiments of the invention, RNAi is accomplished byexpressing an inhibitory sequence that comprises a first polynucleotidesequence containing the nucleotides from about position 1420 to aboutposition 1580 of a cytochrome P450 coding sequence disclosed herein anda sequence that is fully or partially complementary thereto. In some ofthese embodiments, the inhibitory sequence comprises as the firstpolynucleotide sequence the nucleotides corresponding to about position1453 to about position 1551 of the P450 coding sequence set forth in SEQID NO:1 and the second sequence is the complement (i.e., antisensesequence) of this first sequence. The inhibitory sequence can optionallycomprise an intron sequence linked between the first and secondsequences. Any intron known to those of skill in the art can be used inthis manner, as noted herein above. Expression of this inhibitorysequence produces a hairpin RNA (or intron-containing hairpin RNA whenthe intron is present) that also interferes with expression of thecytochrome P450 polypeptides disclosed herein.

The expression cassette for hpRNA interference may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,it is the loop region that determines the specificity of the RNAinterference. See, for example, WO 02/00904, herein incorporated byreference.

Transcriptional gene silencing (TGS) may be accomplished through use ofhpRNA constructs wherein the inverted repeat of the hairpin sharessequence identity with the promoter region of a gene to be silenced.Processing of the hpRNA into short RNAs that can interact with thehomologous promoter region may trigger degradation or methylation toresult in silencing (Aufsatz et al. (2002) Proc. Natl. Acad. Sci. 99(Suppl. 4):16499-16506; Mette et al. (2000) EMBO J. 19(19):5194-5201).

In further embodiments, a polynucleotide may be utilized that encodes azinc finger protein that binds to a gene encoding a cytochrome P450polypeptide, resulting in reduced expression of the gene. In particularembodiments, the zinc finger protein binds to a regulatory region of acytochrome P450 polypeptide gene. In other embodiments, the zinc fingerprotein binds to a messenger RNA encoding a cytochrome P450 polypeptideand prevents its translation. Methods of selecting sites for targetingby zinc finger proteins have been described, for example, in U.S. Pat.No. 6,453,242, and methods for using zinc finger proteins to inhibit theexpression of genes in plants are described, for example, in U.S. PatentPublication No. 20030037355; each of which is herein incorporated byreference.

In other embodiments of the invention, the polynucleotide encodes anantibody that binds to at least one cytochrome P450 polypeptide, andreduces the activity of a cytochrome P450 polypeptide of the presentinvention. In another embodiment, the binding of the antibody results inincreased turnover of the antibody-cytochrome P450 polypeptide complexby cellular quality control mechanisms. The expression of antibodies inplant parts and the inhibition of molecular pathways by expression andbinding of antibodies to proteins in plant parts are well known in theart. See, for example, Conrad and Sonnewald (2003) Nature Biotech.21:35-36, incorporated herein by reference.

In other embodiments, the activity of a cytochrome P450 polypeptide ofthe present invention is reduced or eliminated by disrupting the geneencoding the cytochrome P450 polypeptide. The gene encoding thecytochrome P450 polypeptide may be disrupted by any method known in theart, for example, by transposon tagging or by mutagenizing plants usingrandom or targeted mutagenesis and selecting for plants that havereduced cytochrome P450 activity.

Transposon tagging may be used to reduce or eliminate the activity ofone or more cytochrome P450 polypeptides of the present invention.Transposon tagging comprises inserting a transposon within an endogenouscytochrome P450 gene to reduce or eliminate expression of the cytochromeP450 polypeptide.

In this embodiment, the expression of one or more cytochrome P450polypeptides is reduced or eliminated by inserting a transposon within aregulatory region or coding region of the gene encoding the cytochromeP450 polypeptide. A transposon that is within an exon, intron, 5′ or 3′untranslated sequence, a promoter, or any other regulatory sequence of acytochrome P450 polypeptide gene may be used to reduce or eliminate theexpression and/or activity of the encoded cytochrome P450 polypeptide.

Methods for the transposon tagging of specific genes in plants are wellknown in the art. See, for example, Maes et al. (1999) Trends Plant Sci.4:90-96; Dharmapuri and Sonti (1999) FEMS Microbiol. Lett. 179:53-59;Meissner et al. (2000) Plant J. 22:265-274; Phogat et al. (2000) J.Biosci. 25:57-63; Walbot (2000) Curr. Opin. Plant Biol. 2:103-107; Gaiet al. (2000) Nucleic Acids Res. 28:94-96; Fitzmaurice et al. (1999)Genetics 153:1919-1928).

Additional methods for decreasing or eliminating the expression ofendogenous genes in plants are also known in the art and can besimilarly applied to the instant invention. These methods include otherforms of mutagenesis, such as ethyl methanesulfonate-inducedmutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesisused in a reverse genetics sense (with PCR) to identify plant lines inwhich the endogenous gene has been deleted. For examples of thesemethods see Ohshima et al. (1998) Virology 243:472-481; Okubara et al.(1994) Genetics 137:867-874; and Quesada et al. (2000) Genetics154:421-436; each of which is herein incorporated by reference. Inaddition, a fast and automatable method for screening for chemicallyinduced mutations, TILLING (Targeting Induced Local Lesions In Genomes),using denaturing HPLC or selective endonuclease digestion of selectedPCR products is also applicable to the instant invention. See McCallumet al. (2000) Nat. Biotechnol. 18:455-457, herein incorporated byreference.

Mutations that impact gene expression or that interfere with thefunction of the encoded cytochrome P450 protein can be determined usingmethods that are well known in the art. Insertional mutations in geneexons usually result in null-mutants. Mutations in conserved residuescan be particularly effective in inhibiting the metabolic function ofthe encoded protein. Conserved residues of plant cytochrome P450polypeptides suitable for mutagenesis with the goal to eliminateactivity of a cytochrome P450 polypeptide in converting nicotine tonornicotine in a Nicotiana plant or plant part have been described (See,for example, FIGS. 3 and 4). Such mutants can be isolated according towell-known procedures.

In another embodiment of this invention, dominant mutants can be used totrigger RNA silencing due to gene inversion and recombination of aduplicated gene locus. See, for example, Kusaba et al. (2003) Plant Cell15:1455-1467.

While a number of sequences are recognized in the practice of theinvention, in particular SEQ ID NO:3 and SEQ ID NO:5 find particularuse. While not bound by any particular mechanisms of action, it isbelieved that these sequences encode a nicotine demethylase thatcatalyzes the oxidative N-demethylation of nicotine to nornicotine.Thus, methods to specifically inhibit these coding sequences and notother P450 sequences may be beneficial to the recombinant plant. Thatis, strategies that would lead to inhibition of gene function of thisindividual locus may prove to be superior to those that inhibit theentire gene family. The P450 enzymes are involved in many mechanisms inthe plant, the inhibition of which may prove deleterious or detrimentalto the growth and development of the plant or may negatively impactfactors such as the disease defense capabilities of the plant. Likewise,because the Nicotiana plant P450 enzymes have been implicated in plantmetabolites such as phenylpropanoid, alkaloids, terpenoids, lipids,cyanogenic glycosides, glucosinolates, and a host of other chemicalentities, disruption of p450 activity may alter components involved intobacco flavor, texture, or other properties that would impact thecommercial usefulness of the plant. Therefore, the use of the methodsdiscussed above to inhibit expression in a manner that specificallytargets the coding sequence of SEQ ID NO:3 or SEQ ID NO:5 may bepreferred, including targeted mutational strategies, such aschimeraplasty. See, for example, Stewart et al. (2000) Biotechniques29(4): 838-843; Graham et al. (2002) Biochim Biophys Acta 1587(1):1-6,herein incorporated by reference.

The protein encoded by the cDNA designated 3D_C12-10 (SEQ ID NO:4)differs from 3D_C12-7 (SEQ ID NO:6) at only two amino acid residuesimmediately following the start methionine. The codons corresponding tothese amino acids were contained within the PCR primer used to generatethe 3D_C12-7 cDNA. Thus, the original mRNA template from which 3D_C12-7was amplified may be the same as that corresponding to the 3D_C12-10gene, with the PCR primer sequences mediating the changes observed inthe second and third amino acid sequence. Regardless, the encodedprotein products would function identically. The location of the twoamino acids that differ between the predicted proteins is in theN-terminal signal sequence that merely serves to anchor the protein tothe endoplasmic reticulum membrane and therefore would not be expectedto influence the catalytic properties of the enzyme.

In another embodiment of the invention, the compositions of theinvention find use in screening methods to identify nonconverter plantsfor use in breeding programs. In this manner, the nucleotide sequencesof the invention can be used to screen native germplasms fornonconverter plants having a stable mutation in one or more p450 genesidentified herein. These nonconverter plants identified by the methodsof the invention can be used to develop breeding lines.

In addition to the nucleotide sequences encoding P450 coding sequences,compositions of the invention include an intron sequence in the3D_C12-10 sequence that can be used in screening methods. While notbound by any mechanism of action, the 3D_C12-7/3D_C12-10 gene(s) mayrepresent the only member(s) of the 3D_C12 family involved in themetabolic conversion of nicotine to nornicotine (and as statedpreviously there is a good likelihood that the 3D_C12-7 and 3D_C12-10cDNAs originated from a single unique genetic locus). For certainapplications it would be useful to have a means of diagnosticallydifferentiating this specific member of the 3D_C12 gene family from therest of the closely related sequences within this family. For example,it is possible that within the naturally existing tobacco germplasm (orin mutagenized populations), accessions may exist in which this gene isnaturally dysfunctional and may therefore may be valuable as apermanently nonconverter resource. A method to specifically assay forsuch genotypes (e.g. deletion mutants, rearrangements, and the like)could serve as a powerful tool. To obtain such a tool, the sequencealignment shown in FIG. 3A-3G was used to design PCR primers in regionspossessing polymorphisms among the members. One primer combination (5′primer shown in SEQ ID NO:25 and 3′ primer shown in SEQ ID NO:26) usingsequences specific to 3D_C12-10 yields two particularly useful results:(1) all PCR products amplified from tobacco genomic DNA gave that sameunique product (as determined by DNA sequence analysis); and (2) thepresence of a 992 bp intron was revealed that is located between theprimer sequences (FIG. 7; intron shown in SEQ ID NO:24).

When any cDNA corresponding to a member of the 3D_C12 family is used asa hybridization probe in a Southern blotting assay of tobacco genomicDNA, a complex pattern is observed. This is expected, given that thereare multiple, closely related members of this gene family. Because theintron regions of genes are typically less conserved than exons, it ispredicted that the use of an intron-specific probe would reduce thiscomplexity and better enable one to distinguish the gene(s)corresponding to the 3D_C12-7/3D_C12-10 gene from the other members ofthe family. Indeed, the probe corresponding to the sequence shown inFIG. 7 resulted in a Southern blotting pattern with greatly reducedcomplexity. The use of a 3D_C12-10 intron-specific probe, and/or the PCRprimers used to generate the fragment shown in FIG. 7, therefore providepowerful tools in assays to determine whether any naturally occurring,or mutagenized, tobacco plants possess deletions or rearrangements thatmay render the gene inactive. Such a plant can then be used in breedingprograms to create tobacco lines that are incapable of converting.

Transformed Plants, Plant Parts, and Products Having Reduced Nornicotineand NNN Content

The cytochrome P450 polynucleotides of the invention, and variants andfragments thereof, can be used in the methods of the present inventionto inhibit expression or function of cytochrome P450s that are involvedin the metabolic conversion of nicotine to nornicotine in a plant. Inthis manner, inhibitory sequences that target expression or function ofa cytochrome P450 polypeptide disclosed herein are introduced into aplant or plant cell of interest. In some embodiments the expressioncassettes described herein are introduced into a plant of interest, forexample, a Nicotiana plant as noted herein below, using any suitabletransformation methods known in the art including those describedherein.

The methods of the invention do not depend on a particular method forintroducing a sequence into a plant or plant part, only that the desiredsequence gains access to the interior of at least one cell of the plantor plant part. Methods for introducing polynucleotide sequences intoplants are known in the art and include, but are not limited to, stabletransformation methods, transient transformation methods, andvirus-mediated methods.

Transformation protocols as well as protocols for introducingheterologous polynucleotide sequences into plants vary depending on thetype of plant or plant cell targeted for transformation. Suitablemethods of introducing polynucleotides into plant cells of the presentinvention include microinjection (Crossway et al. (1986) Biotechniques4:320-334), electroporation (Shillito et al. (1987) Meth. Enzymol.153:313-336; Riggs et al. (1986) Proc. Natl. Acad. Sci. USA83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. Nos.5,104,310, 5,149,645, 5,177,010, 5,231,019, 5,463,174, 5,464,763,5,469,976, 4,762,785, 5,004,863, 5,159,135, 5,563,055, and 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), andballistic particle acceleration (see, for example, U.S. Pat. Nos.4,945,050, 5,141,131, 5,886,244, 5,879,918, and 5,932,782; Tomes et al.(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988)Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann. Rev.Genet. 22:421-477; Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues,ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppleret al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992)Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation);D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); all ofwhich are herein incorporated by reference.

Any plant tissue that can be subsequently propagated using clonalmethods, whether by organogenesis or embryogenesis, may be transformedwith a recombinant construct comprising a cytochrome P450 inhibitorysequence, for example, an expression cassette of the present invention.By “organogenesis” in intended the process by which shoots and roots aredeveloped sequentially from meristematic centers. By “embryogenesis” isintended the process by which shoots and roots develop together in aconcerted fashion (not sequentially), whether from somatic cells orgametes. Exemplary tissues that are suitable for various transformationprotocols described herein include, but are not limited to, callustissue, existing meristematic tissue (e.g., apical meristems, axillarybuds, and root meristems) and induced meristem tissue (e.g., cotyledonmeristem and hypocotyl meristem), hypocotyls, cotyledons, leaf disks,pollen, embryos, and the like.

As used herein, the term “stable transformation” is intended to meanthat the nucleotide construct of interest introduced into a plantintegrates into the genome of the plant and is capable of beinginherited by the progeny thereof “Transient transformation” is intendedto mean that a sequence is introduced into the plant and is onlytemporally expressed or is only transiently present in the plant.

In specific embodiments, the inhibitory sequences of the invention canbe provided to a plant using a variety of transient transformationmethods. The inhibitory sequences of the invention can be transientlytransformed into the plant using techniques known in the art. Suchtechniques include viral vector systems and the precipitation of thepolynucleotide in a manner that precludes subsequent release of the DNA.Thus, the transcription from the particle-bound DNA can occur, but thefrequency with which it is released to become integrated into the genomeis greatly reduced. Such methods include the use of particles coatedwith polyethyenlimine (PEI; Sigma #P3143).

In other embodiments, the inhibitory sequence of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anexpression cassette of the invention within a viral DNA or RNA molecule.It is recognized that promoters for use in the expression cassettes ofthe invention also encompass promoters utilized for transcription byviral RNA polymerases. Methods for introducing polynucleotides intoplants and expressing a protein encoded therein, involving viral DNA orRNA molecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al.(1996) Molecular Biotechnology 5:209-221; herein incorporated byreference.

Transformed cells may be grown into Nicotiana plants in accordance withconventional methods. See, for example, methods disclosed in Vasil andHildebrandt (1965) Science 150:889; Negaard and Hoffman (1989)Biotechniques 7(8):808-812. These plants may then be grown, and eitherpollinated with the same transformed line or different lines, and theresulting progeny having expression of the desired phenotypiccharacteristic identified, i.e., reduced expression of one or morecytochrome P450s that are involved in the metabolic conversion ofnicotine to nornicotine, and thus reduced content of nornicotine, and aconcomitant reduced content of its nitrosamine metabolite, NNN, in theplant, particularly in the leaf tissues. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

The compositions and methods of the invention can be used to reduce thenornicotine content, particularly in the leaves and stems, of any plantof the genus Nicotiana including, but not limited to, the followingspecies: acuminata, affinis, alata, attenuate, bigelovii, clevelandii,excelsior, forgetiana, glauca, glutinosa, langsdorffii, longiflora,obtusifolia, palmeri, paniculata, plumbaginifolia, qudrivalvis, repanda,rustica, suaveolens, sylvestris, tabacum, tomentosa, trigonophylla, andx sanderae. The present invention also encompasses the transformation ofany varieties of a plant of the genus Nicotiana, including but notlimited to Nicotiana acuminata multiflora, Nicotiana alata grandiflora,Nicotiana bigelovii quadrivalvis, Nicotiana bigelovii wallacei,Nicotiana obtusifolia obtusifolia, Nicotiana obtusifolia plameri,Nicotiana quadrivalvis bigelovii, Nicotiana quadrivalvis quadrivalvis,Nicotiana quadrivalvis wallacei, and Nicotiana trigonophylla palmeri, aswell as varieties commonly known as flue or bright varieties, Burleyvarieties, dark varieties, and oriental/Turkish varieties.

The transgenic plants of the genus Nicotiana as described herein aresuitable for conventional growing and harvesting techniques, such ascultivation in manure rich soil or without manure, bagging the flowersor no bagging, or topping or no topping. The harvested leaves and stemsmay be used in any traditional tobacco product including, but notlimited to, pipe, cigar and cigarette tobacco, and chewing tobacco inany form including leaf tobacco, shredded tobacco, or cut tobacco.

Thus the present invention provides a Nicotiana plant, particularly leaftissues of these plants, comprising an expression cassette of theinvention and a reduced amount of nornicotine and N′-nitrosonornicotine.As used herein, the term “a reduced amount” or “a reduced level” isintended to refer to an amount of nornicotine and/orN′-nitrosonornicotine in a treated or transgenic plant of the genusNicotiana or a plant part or tobacco product thereof that is less thanwhat would be found in a plant of the genus Nicotiana or a plant part ortobacco product from the same variety of tobacco, processed (i.e.,cultured and harvested) in the same manner, that has not been treated orwas not made transgenic for reduced nornicotine and/orN′-nitrosonornicotine. The amount of nornicotine may be reduced by about10% to greater than about 90%, including greater than about 20%, about30%, about 40%, about 50%, about 60%, about 70%, and about 80%.

The term “tobacco products” as used herein include, but are not limitedto, smoking materials (e.g., cigarettes, cigars, pipe tobacco), snuff,chewing tobacco, gum, and lozenges. The present invention alsoencompasses a range of tobacco product blends that can be made bycombining conventional tobacco with differing amounts of the lownornicotine and/or N′-nitrosonornicotine tobacco described herein. Infurther embodiments, the plant or plant part of the genus Nicotiana asdescribed above is cured tobacco.

In some embodiments of the present invention, the tobacco productreduces the carcinogenic potential of tobacco smoke that is inhaleddirectly with consumption of a tobacco product such as cigars,cigarettes, or pipe tobacco, or inhaled as secondary smoke (i.e., by anindividual that inhales the tobacco smoke generated by an individualconsuming a tobacco product such as cigars, cigarettes, or pipetobacco). The cured tobacco described herein can be used to prepare atobacco product, particularly one that undergoes chemical changes due toheat, comprising a reduced amount of nornicotine and/orN′-nitrosonornicotine in the smoke stream that is inhaled directly orinhaled as secondary smoke. In the same manner, the tobacco products ofthe invention may be useful in the preparation of smokeless tobaccoproducts such as chewing tobacco, snuff, and the like.

The tobacco products obtained from the transgenic tobacco plants of thepresent invention thus find use in methods for reducing the carcinogenicpotential of these tobacco products, and reducing the exposure of humansto the carcinogenic nitrosamine NNN, particularly for individuals thatare users of these tobacco products.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

The following materials and protocols were utilized in the experimentsdescribed herein below.

Plant Materials

All plant materials utilized in these experiments were provided by Dr.Earl Wernsman, Department of Crop Science, North Carolina StateUniversity. DH 91-1307-46 (NC) and DH91-1307-46 (Con) are near-isogenicdoubled haploid Burley lines (nonconverter and converter, respectively)recovered from the same maternal haploid plant. Burley lines DH 98-326-3(nonconverter) and DH 98-326-1 (converter), and DH 98-325-5(nonconverter) and DH 98-325-6 (converter) represent two additionalpairs of near-isogenic lines. SC58 is a flue-cured tobacco variety,nonconverter individuals of which are designated SC58(c_(T)c_(T)).SC58(C_(T)C_(T)) is a near-isogenic stable converter line thatoriginated though the introgression of the single dominant converterlocus (C_(T)) found in the tobacco progenitor species N. tomentosiformisinto SC58 (Mann et al. (1964) Crop Sci. 4:349-353. After eightadditional backcrosses to SC58, the near-isogenic SC58(C_(T)C_(T)) linewas created and subsequently maintained via self-fertilization.

All plants were maintained in growth chambers or greenhouses usingstandard potting soil and fertilizer. For the microarray studies, themetabolism of nicotine to nornicotine was accelerated by excisingindividual leaves and inserting their petioles into a solution of 0.1%ethephon or 1% sodium bicarbonate. The leaves were then placed in agrowth chamber (27° C.) for 5 to 7 hours to facilitate the entry of theethephon or sodium bicarbonate solutions throughout the transpirationalstream. The treated leaves were placed in small plastic storage bagsafter being lightly sprayed with water (to maintain high humidity) andcured for three days at 30° C. in the dark. To enhance the nicotine tonornicotine conversion in the transgenic plants generated in this study,detached leaves were dipped into a solution of 0.2% ethephon, dried, andcured in plastic storage bags for seven days at room temperature in thedark.

cDNA Libraries and Expressed Sequence Tags

Total cellular RNA was isolated from senescing leaf tissue of Burleylines DH 91-1307-46 (NC) and DH 91-1307-46 (Con) using the TRIzol®reagent according to the manufacturer's protocol (Invitrogen). PolyA+RNA was isolated from total RNA using the MessageMaker system(Invitrogen), and cDNA was subsequently synthesized and cloned into thelambda ZAP II phage vector using the ZAP-cDNA Synthesis and Gigapack IIIGold Cloning Kit (Stratagene). Aliquots of the phage libraries wereconverted to pBluescript-based plasmid libraries following the massexcision protocol outlined by Stratagene.

Thousands of colonies from both the converter and nonconverter librarieswere grown on selective solid media and picked into 384-well platescontaining Luria broth (with ampicillin) in 10% glycerol to facilitatelong term storage of the clones at −80° C. Over 11,000 clones from eachlibrary were transferred from the 384-well plates to 96-well growthblocks and grown in selective media. Plasmids were isolated in 96-wellformat using the R.E.A.L. Preparation Kit (Qiagen) with the aid of aBioRobot 3000 Workstation (Qiagen). To generate the ESTs, the plasmidclones were sequenced using the T3 primer (Qiagen) and BigDye®Terminator system (Applied Biosystems) according to the BigDye® cyclesequencing protocol. Performa® DTR 96-well plates (Edge Biosystems) wereused to remove the unincorporated dye from the sequencing reactionsprior to loading the samples onto a Perkin Elmer Prism 3700 96-CapillaryAutomated DNA Sequencer.

Preparation of DNA Chips

To obtain DNAs suitable for spotting onto glass slides, the M13 forwardand reverse sequencing primers (Qiagen) were used as PCR primers toamplify cDNA inserts from the plasmids containing cDNAs represented inthe EST databases. Plasmid clones were subjected to PCR in 96-wellformat using an Applied Biosystems Gene Amp 9700 model thermocycler. Theresulting PCR products were processed through Millipore Multiscreen™ PCRor Montage™ PCRμ96 purification systems. The resulting products weretransferred into 384-well plates containing equal volumes of DSMO. Thefinal DNA concentrations were estimated to be equal to or greater than0.1 mg/ml. The DNAs were subsequently spotted onto amino silane-coatedslides (Corning® GAPS II) using an Affymetrix GMS 417 array printer.DNAs were immobilized to the slide surface by UV crosslinking (˜120mJ/m²), followed by baking at 75° C. for two hours.

Microarray Hybridization and Analysis

The amino allyl dUTP-based indirect method of dye incorporationdescribed by “The Institute of Genome Research”(http://pga.tigr.org/protocols.html) was used to label nonconverter andconverter RNAs with Cy3 and Cy3 fluorescent dyes (Amersham Biosciences).Briefly, 20 μg of total RNA was reverse transcribed in a 30 μl volumecontaining 400 units of SuperScript II RT (Invitrogen), 6 ng randomhexamer primers, 0.5 mM each of dATP, dCTP, and dGTP, 0.3 mM dTTP, and0.2 mM amino allyl dUTP (Sigma) in first strand synthesis buffer(Invitrogen). Reactions were incubated for 6 to 14 hours at 42° C.,followed by hydrolysis of the RNA with NaOH. The resulting first strandcDNA molecules were column purified (Qiagen) and washed with phosphatebuffer. Coupling reactions of the NETS-ester Cy3 or Cy5 fluorescent dyesto the cDNA occurred during incubation in 0.05 M sodium carbonate buffer(pH 9.0) and 25% DMSO at room temperature for 1.5 hours.

Microarray slides were prehybridized in a solution of 5×SSC, 0.1% SDS,and 1% BSA at 42° C. for 45 minutes, rinsed gently with dH₂O andisopropanol, and dried by low speed centrifugation. The Cy3- andCy5-labeled cDNAs were column purified (Qiagen), combined, andhybridized to the DNA slides in a solution containing 5×SSC, 0.5% SDS,5× Denhardt's, 0.45 μg/μl Poly A RNA, 0.45 μg/μl calf thymus DNA, and50% formamide. The slides were incubated with the hybridization solutionfor 14 to 16 hours at 42° C. Post-hybridization washes consisted ofsequential 4-minute incubations with the following solutions: 1×SSC,0.2% SDS; 0.1×SSC, 0.2% SDS; 0.1×SSC, and a final 10 second rinse with0.01×SSC.

The microarrays were subsequently scanned using ScanArray 2.1 (GSILumonics) or ScanArray Express (PerkinElmer). Sequential scanning forCy5 and Cy3 fluorescence was performed at a maximal resolution of 10gm/pixel, and laser power and PMT gain adjusted to provide reliable andequivalent signal strengths. The acquired array images were quantifiedfor signal intensity with QuantArray™ analysis software (PerkinElmer),using the histogram-based method. Total intensities were used asquantification output fields, and the acquired data sets were saved asUnicode, tab-delimited text files. Importation of the text files intoMicrosoft Excel enabled the subsequent calculation of Cy5/Cy3 andCy3/Cy5 ratios, the statistic we employed for the identification ofcandidate genes.

Cloning Full-Length and Additional Members of the 3DC12 Gene Family

To clone the entire coding region of 3D_C12 and 7D_A06 a modified5′-RACE strategy was employed using a pBluescript II vector-specificforward primer (BlueSK; 5′-CGCTCTAGAACTAGTGATC-3′; SEQ ID NO:17) and aset of gene-specific reverse primers. Two 3D_C12-specific reverseprimers were designed, one of which is complementary to the downstreamportion of the 3′ untranslated region (5′TTTTTGGGACAATCAGTCAA-3′; SEQ IDNO:18) and the other complementary to a sequence within the codingregion (5′-GTTAGATTTATCGTACTCGAATT-3′; SEQ ID NO:19). For the formerprimer, the first five Ts are complementary to the polyA tail of thetranscript. A 7D_A06-specific reverse primer(5′-TTCATTTCAAATTATTTTATGCACCA-3; SEQ ID NO:20) was also designed, andis complementary to a segment in the 3′ untranslated region of thisgene. PCR reactions contained 10 ng of converter tobacco leaf cDNAlibrary (within the pBluescript vector) as template, 2 μM concentrationof each primer, 350 μM of each dNTP, and 1.5 mM MgCl₂ in a finalreaction volume of 50 μL. Amplification was initiated by the addition of2.5 units of UniPol enzyme mix using conditions described by themanufacturer (Roche). After an initial denaturation step at 94° C. for 4minutes, the samples were subjected to 30 cycles of denaturation at 94°C. for 15 seconds, annealing at 57° C. for 30 seconds, and extension at72° C. for 90 seconds. A final extension step at 72° C. for 10 minuteswas included at the end of the 30 cycles. The amplicons were ligatedinto the pGEM Easy T/A vector (Promega), and 10 randomly selected clonesfrom each amplification were subjected to DNA sequence analysis. Nucleicacid and predicted protein sequences of the various members of the3D_C12 gene family were analyzed and compared using the BLASTX (Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402), ClustalW (Higgins et al.(1994) Nucleic Acids Res. 22:4673-4680) and GAP (University of WisconsinGenetic Computing Group software package) algorithms.

The above described strategy was effective in identifying full-lengthsequence information for 3D_C12 and 7D_A06. In addition, PCRamplifications using the PCR primer internal to the 3D_C12 coding regiongave rise to partial-sequence information for the unique 3D_C12-15 cDNA.In an attempt to obtain full-length sequence information for 3D_C12-15,a gene-specific primer complementary to the 5′ terminus of its codingregion (5′-ATGGTTTTTCCCATAGAAGCC-3′; SEQ ID NO:21) was used inconjunction with a pBluescript-specific reverse primer(5′-TCGAGGTCGACGGTATC-3; SEQ ID NO:22). Although a full-length 3D_C12-15cDNA was not recovered, this amplification resulted in the isolation of3D_C12-7, which proved to be another unique member of the 3D_C12 genefamily.

Transgenic Plant Analysis

The RNAi-based gene silencing constructs were assembled in a version ofthe pKYL80 cloning vector (Schardl et al. (1987) Gene 61:1-11) that wasengineered to contain a 151-bp fragment of the soybean FAD3 gene intronbetween the Xhol and SacI restriction sites of the polylinker(pKYLX80I). To create a construct in which the FAD3 intron was flankedby a sense and antisense fragment of 3D_C12, a 99-bp region locatedimmediately upstream of the stop codon of the 3D_C12 cDNA (FIG. 3A-3G)was cloned between the HindIIl-Xhol and SacI-Xbal restriction sites ofpKYLX80I in its sense and antisense orientation, respectively. Theresulting HindIII-Xbal fragment containing the 3D_C12 sense arm, FAD3intron, and 3D_C12 antisense arm was subcloned into the pKYLX71 plantbinary expression vector (Maiti et al. (1993) Proc. Natl. Acad. Sci. USA90:6110-6114) between the 35S CaMV promoter and a rubisco small subunitterminator.

Overexpression constructs were created by replacing the 3-glucuronidaseORF of the plant binary expression vector pBI121 (Clontech) with thefull-length coding regions of the 3D_C12, 7D_A06, and 3D_C12-7 cDNAs.This placed the tobacco P450s under the transcriptional control of the35S CaMV promoter. The pBI121- and pKYLX71-based constructs weretransformed into Agrobacterium tumefaciens strain LBA 4404 andintroduced into tobacco cultivars Petite Havana and DH98-325-6(converter), respectively, using established protocols (Horsch et al.(1985) Science 227:1229-1231).

Northern Blot Analysis

Total cellular RNAs were isolated from tobacco leaves using the TRIZOL®method as described by the manufacturer (Invitrogen). Five to tenmicrograms of RNA were size fractionated on a 1.2% agarose gel preparedin TBE buffer. RNA immobilization, probe labeling, and signal detectionwere carried out using the DIG nucleic acid labeling and detection kitsaccording to the manufacturer's instructions (Roche). Alternatively,probes were synthesized using ³²P-dCTP according to protocolsaccompanying the Random Primed DNA Labeling kit (Roche).

Alkaloid Analysis

Tobacco leaves were harvested and air dried in an oven at 65° C. for 2days. A 100 mg sample of crushed, dried leaf was added to 0.5 ml of 2 NNaOH in a 20 mL scintillation vial. The sample was mixed and allowed toincubate for 15 minutes at room temperature. Alkaloids were extracted bythe addition of 5 mL of extraction solution [0.04% quinoline (wt/vol)dissolved in methyl-t-butyl ether] and gently rotated on a linear shakerfor 3 hours. Following phase separation, an aliquot of the organic phasewas transferred to a sample vial. Samples were analyzed using aPerkinElmer Autosystem XL gas chromatograph equipped with a flameionization detector, a 4 mm split/splitless glass liner, and a 30 m×0.53mm ID DB-5 column. Chromatographic conditions were as follows: detectortemperature: 250° C.; injector temperature: 250° C.; helium flow rate at120° C.: 20 mL/min; injection volume: 2 μL; column conditions: 120° C.,hold 1 minute, 120-280° C. at 30° C. /minute ramping rate, hold at 280°C. for 2 minutes. Alkaloid composition was determined by the TotalChromeNavigator software using a calibration curve.

EXAMPLE 1 Generation of EST Databases

RNAs isolated from senescing leaves of the converter genotype DH91-1307-46 (Con) and its near-isogenic nonconverter counterpart DH91-1307-46 (NC) were used to generate cDNA libraries. High-throughputautomated DNA sequencers were initially used to generate single-runsequence information (ESTs) for 11,136 randomly chosen cDNAs from theconverter library. The local alignment search tool BLASTX (Altschul etal. (1990) J. Mol. Biol. 215:403-410) was used to compare the predictedprotein sequence of each tobacco cDNA with the nonredundant proteindatabase curated by the National Center for Biotechnology Information ofthe National Library of Medicine and National Institutes of Health.Subsequently, a similar annotated EST database was generated byconducting sequencing runs on 11,904 cDNAs selected from thenonconverter library.

EXAMPLE 2 Microarray Analyses of Converter CDNA Library Methods

Upon completion of the EST database generated from the converter cDNAlibrary, the inserts from 4992 clones were amplified by PCR and spottedonto glass slides. Given the possibility that the nicotine demethylaseenzyme may be catalyzed by an enzyme of the P450 class of oxidativeenzymes, special attention was given to library entries that werepredicted by BLASTX analysis to encode P450s.

From visual inspection of the BLASTX results, it was estimated that 31unique P450 genes were represented in the database. When selectingspecific 96-well plates to be included on the microarray, care was takento ensure that all unique P450 genes would be included among the 4992cDNAs selected.

RNAs isolated from the near-isogenic Burley genotypes DH 98-326-3(nonconverter) and DH 98-326-1 (converter), and DH 98-325-5(nonconverter) and DH 98-325-6 (converter) were used to generate Cy3-and Cy5-labeled cDNAs. To maximize the metabolic conversion of nicotineto nornicotine in converter genotypes, detached leaves were treated withsodium bicarbonate or ethephon prior to curing, treatments that havebeen shown to accelerate nornicotine production in converter plantswhile having no effect in nonconverter individuals (Fannin and Bush(1992) Med. Sci. Res. 20:867-868; Shi et al. (2003) J. Agric. Food Chem.51:7679-7683).

To minimize the variability inherent with microarray experiments,reciprocal experiments were conducted simultaneously. In this manner, DH98-325-5 RNA was labeled with Cy5 and DH 98-325-6 RNA was labeled withCy3, and then in a reciprocal experiment DH 98-325-5 RNA was labeledwith Cy3 and DH 98-325-6 RNA was labeled with Cy5 (collectively referredto as Exp. 2.1). Similarly, DH 98-326-3 and DH 98-326-1 RNAs werelabeled with Cy3 and Cy5, respectively, in one experiment, and then thesame RNAs were labeled with Cy5 and Cy3, respectively, in a reciprocalexperiment (collectively referred to as Exp. 2.2).

Even when conducted reciprocally, the results of any given microarrayexperiment are likely to include “false positives,” representing genesthat are differentially regulated between a specific genotypic pairand/or uniquely in response to a specific treatment, as opposed todifferences directly associated with the conversion phenomenon. Todefine the set of candidate genes that are most likely to be upregulateddue to the conversion process, cDNAs were identified that met thefollowing criteria: for any set of reciprocal experiments (i.e., Exp.2.1, or Exp. 2.2), the hybridization intensity of a given cDNA had to beat least 2-fold higher with the converter probe than nonconverter probein at least one of the hybridizations, and not less than 1.5-fold higherin the reciprocal experiment.

Experiment 2.1—Leaves from near-isogenic lines DH 98-325-5 and DH98-325-6 were treated with ethephon and cured for 3 days at 30° C.Alkaloid analysis revealed that virtually all of the nicotine had beenmetabolized to nornicotine in the DH 98-325-6 leaf during this periodwhile minimal nornicotine was observed in the DH 98-325-5 leaf. RNAsfrom the DH 98-325-5 nonconverter plant were labeled with the Cy3fluorescent dye, and RNAs extracted from a DH 98-325-6 (converter) leafwere labeled with Cy5. The Cy3- and Cy5-labeled cDNAs were incubatedtogether on the same DNA chip and allowed to hybridize overnight.

Experiment 2.2—A microarray analysis similar to Exp. 2.1 was conductedusing the DH 98-326-3 (nonconverter) and DH 98-326-1 (converter)near-isogenic lines. In these experiments, leaves from each genotypewere treated with 1% sodium bicarbonate and cured for 3 days at 30° C.At the end of the treatment period, nicotine was the predominantalkaloid in the DH 98-326-3 leaf, while nearly all of the alkaloid inthe DH 98-326-1 leaf was nornicotine. As described for Exp. 2.1, theseexperiments were reciprocally conducted.

Results

In both Experiment 2.1 and Experiment 2.2, the great majority of the4992 cDNAs spotted on the glass slides showed no substantial differencesin their hybridization intensities to the competing Cy3- and Cy5-labeledprobes.

Of the 4992 cDNAs spotted on the glass slides, only five showed at least2-fold higher expression in one hybridization and not less than 1.5-foldin the reciprocal hybridization for both Exp. 2.1 and Exp. 2.2. Theseentries were designated 3D_C12, 7D_A06, 27C_C12, 33A_D06, and 34D_F06.BLASTX analysis of the partial sequence information for 3D_C12 and7D_A06 found in our EST database predicted that the cDNAs encode twoclosely related P450 enzymes. 27C_C12 and 33A_D06 were predicted toencode glycine-rich cell wall proteins, displaying over 90% sequenceidentity to small tobacco glycine-rich proteins found in GenBank (e.g.,Accession No. AAK57546). Clone 34D_F06 was found to contain a doublecDNA insert, one insert showing homology to serine/threonine proteinkinases, and the other showing high sequence identity to the sameglycine-rich cell wall proteins as the 27C_C12 and 33A D06 cDNAs.

EXAMPLE 3 Microarray Analysis of CDNA Non-Converter Library

Upon completion of the EST database from the nonconverter library(generated from senescing leaves of genotype DH 91-1307-46 (NC)),another set of microarray experiments was initiated. For this nextgeneration of microarrays, the goal was to produce glass slidescontaining the complete nonredundant set of genes represented in bothlibraries.

To obtain an estimate of the number of unique genes that are representedin the database, clustering analysis was conducted to identify ESTspredicted to be represented multiple times in the database (contigs)versus those predicted to be represented only once (singletons) (Huangand Madan (1999) Genome Res. 9:868-877). Due to the nature of theclustering algorithms, sequences showing high, but imperfect, sequenceidentities are clustered into the same contig. The total set ofpredicted unique genes, or unigenes, within a database is calculated asthe sum of the contigs and singletons. Clustering analysis of thecombined converter and nonconverter databases predicted 2246 contigs and4717 singletons for a total of 6,963 unigenes. Inserts from allsingletons and an individual from each contig were amplified by PCR andspotted onto glass slides, resulting in a gene chip containing thecomplete 6,963 unigene set.

In addition to creating a new DNA chip, the genetic materials used togenerate hybridization probes also differed from those used in Example2. SC58 is a flue-cured tobacco variety, nonconverter individuals ofwhich are designated SC58(c_(T)c_(T)). SC58(C_(T)C_(T)) is anear-isogenic stable converter line that originated though theintrogression of the single dominant converter locus (C_(T)) found inthe tobacco progenitor species N. tomentosiformis into SC58 (Mann et al.(1964) Crop Sci. 4:349-353). After eight additional backcrosses to SC58,the near-isogenic SC58(C_(T)C_(T)) line was created and subsequentlymaintained via self-fertilization. The conversion phenotype ofSC58(C_(T)C_(T)) plants is unique with respect to standard convertertobacco lines in that the metabolism of nicotine to nornicotine in theleaf does not require senescence or curing. Plants possessing the C_(T)converter locus from N. tomentosiformis contain nornicotine as thepredominant alkaloid even in green leaf tissue (Wernsman and Matzinger(1968) Tob. Sci. 12:226-228).

RNAs isolated from green leaf tissue of SC58(c_(T)c_(T)) andSC58(C_(T)C_(T)) were labeled with Cy3 and Cy5, respectively, andsimultaneously hybridized to a DNA chip containing the entire 6,963unigene set of cDNAs. The fluorescent dyes were reversed to produce theprobes for a reciprocal experiment as described in Exps. 2.1 and 2.2 ofExample 2.

Results

Results were evaluated using the same criteria as in Example 2, i.e.,individual cDNAs were identified that showed at least 2-fold enhancedhybridization to the labeled SC58(C_(T)C_(T)) versus SC58(c_(T)c_(T))cDNAs in one experiment and at least 1.5-fold enhancement in thereciprocal assay.

Results were compared to those from Exp. 2.1 and Exp. 2.2 in Example 2above. Enhanced hybridization of converter RNAs to cDNAs encodingmembers of the same closely related P450 family was the only resultshared by all three microarray experiments using the defined criteria.131 A_A02 is the name of the cDNA that was spotted onto the 6963-memberunigene chip that is representative of the closely-related P450 genefamily that includes 3D_C12 and 7D_A06 (3D_C12 and 7D_A06 themselveswere not spotted on the unigene slide). No other cDNAs on the array inExample 3, which included representatives of the contigs containing theglycine-rich protein-encoding 27C_C12 and 33A_D06 and 34D_F06 cDNAs,scored positive by the defined criteria and also scored positive in Exp.2.1 or Exp. 2.2 of Example 2 above, regardless of whether the resultswere compared individually or collectively.

TABLE 1 Microarray results of members of the 3D_C12 gene familyExperiment 2.1 Experiment 2.1 (reciprocal) Cy3 Cy5/Cy3 Cy3 Cy5/Cy3 cDNAreading Cy5 reading ratio reading Cy5 reading ratio 3D_C12 15514.1425928.95 1.67 19355.85 9507.87 2.04 7D_A06 15238.23 37196.19 2.4413651.03 8121.04 1.68 Experiment 2.2 Experiment 2.2 (reciprocal) Cy3Cy5/Cy3 Cy3 Cy5/Cy3 reading Cy5 reading ratio reading Cy5 reading ratio3D_C12 12756.43 28669.28 2.25 32198.81 16166.13 1.99 7D_A06 7571.0619180.94 2.53 42408.85 18440.17 2.30 Example 3 Example 3 (reciprocal)Cy3 Cy5 Cy5/Cy3 Cy3 Cy5/Cy3 reading reading ratio reading Cy5 readingratio 131A_A02 11138.96 19638.82 1.76 36963.45 10085.25 3.67

Combined Results

The combined results of microarray experiments described above definedmembers of a closely related P450 gene family, hereafter referred to asthe 3D_C12 family, to be the best candidates for playing a direct rolein the metabolic conversion of nicotine to nornicotine in convertertobacco plants. The hybridization results of the members of this P450family in each of the three microarray experiments are shown in Table 1.The results of the microarrays were independently confirmed usingNorthern blotting assays. As shown in FIG. 2, an approximately 2-foldhigher signal was observed in senescing, cured converter leaves comparedto their nonconverter counterparts when RNA blots were incubated with aradiolabeled 7D_A06 hybridization probe.

EXAMPLE 4 Sequence Analysis of the 3D_C12 Gene Family

Once microarray experiments defined 3D_C12 and 7D_A06 as potentiallybeing involved in the conversion process, obtaining complete DNAsequence information for these genes became the next step in theircharacterization. The original 3D_C12 and 7D_A06 clones that weresequenced when generating the EST database described elsewhere herein(and spotted onto the microarrays) were not full-length cDNAs. To obtaina full-length sequence, primers were generated corresponding to regionsin the 3′ flanking region and in the interior of the coding regions thatwere sufficiently polymorphic to distinguish between 7D_A06 and 3D_C12.These gene-specific primers were used in combination with primersspecific to the cloning site of pBluescript II to amplify cDNAs from theconverter cDNA library in an attempt to obtain sequence that wouldinclude the complete 5′ ends of the 3D_C12 and 7D_A06 reading frames.

This strategy led to the determination of the DNA sequence correspondingto the complete coding regions of 3D_C12 (nt 1-1551 of SEQ ID NO:1;predicted amino acid sequence shown in SEQ ID NO:2) and 7D_A06 (nt1-1554 of SEQ ID NO:7; predicted amino acid sequence shown in SEQ IDNO:8) (FIGS. 3A-3G and 4). GAP analyses of the 3D_C12 and 7D_A06 DNA andpredicted protein sequences showed that they share 93.4% DNA sequenceidentity and 92.3% identity at the protein level (Tables 2 and 3).Initial BLASTX analysis against the nonredundant GenBank databaserevealed that 3D_C12 and 7D_A06 share greatest sequence homology toCYP82E1, a tobacco P450 gene of unknown function that is upregulated inresponse to fungal elicitors (Takemoto et al. (1999) Plant Cell Physiol.40:1232-1242). The CYP82E1 protein is 66.9% and 67.5% identical to thepredicted amino acid sequences of 3D_C12 (SEQ ID NO:2) and 7D_A06 (SEQID NO:8), respectively, and the CYP82E1 DNA sequence is 72.1% and 73.5%identical to the respective coding sequences for 3D_C12 (nt 1-1551 ofSEQ ID NO:1) and 7D_A06 (nt 1-1554 of SEQ ID NO:7).

TABLE 2 Nucleotide sequence identities between members of the 3D_C12gene family. 3D_C12- 3D_C12- 3D_C12- 3D_C12 7D_A06 7 10 15* 7D_A0693.4** 3D_C12-7 93.7 94.0 3D_C12-10 93.7 94.4 99.7 3D_C12- 95.5 92.693.1 92.8 15* 131A_A02* 98.0 94.0 93.4 93.1 93.1 *partial sequences**numbers indicate percentages

TABLE 3 Predicted amino acid sequence identities between full-lengthmembers of the 3D_C12 gene family. 3D_C12 7D_A06 3D_C12-7 7D_A06 92.3*3D_C12-7 92.8 94.8 3D_C12-10 92.5 94.4 99.6 **numbers indicatepercentages

In addition to enabling the acquisition of full-length sequenceinformation for the 3D_C12 and 7D_A06 cDNAs, the above described PCRamplifications yielded additional products that were closely related to,yet clearly distinct from, the 3D_C12 and 7D_A06 cDNA sequences. Using aprimer directed against a sequence interior to the 3D_C12 cDNA, incombination with a primer specific to pBluescript II, a unique sequencedesignated 3D_C12-15 (FIG. 3A-3G; SEQ ID NO:9; predicted amino acidsequence shown in SEQ ID NO:10) was amplified in addition to theexpected 3D_C12 product. 3D_C12-15 is 95.5% identical to thecorresponding DNA sequence of 3D C12 and 92.6% identical to the sameregion of 7D_A06 (Table 2).

Because the 3D_C12-15 fragment represented an additional, distinctmember of the 3D_C12 gene family, an attempt was made to obtain afull-length cDNA sequence of this gene. A PCR primer specific to thefirst seven codons of the 3D_C12-15 reading frame was used incombination with a pBluescript II-specific primer in an amplificationreaction using our converter cDNA library as template. Sequence analysisof several independent amplification products failed to reveal afull-length 3D_C12-15 gene. Instead, a new member of this family wasrecovered, designated 3D_C12-7 (FIG. 3A-3G; coding sequence set forth asnt 1-1551 of SEQ ID NO:5). Across the full-length nucleotide sequenceshown in SEQ ID NO:5, 3D_C12-7 shares 93.7% nucleotide sequence identitywith 3D_C12 (across SEQ ID NO:1), 94.0% nucleotide sequence identitywith 7D_A06 (across SEQ ID NO:7), and 93.1% identity over thecorresponding region of fragment 3D_C12-15 (SEQ ID NO:9) (Table 2). Thepredicted amino acid sequence of 3D_C12-7 (SEQ ID NO:6) is 92.8%identical to the 3D_C12 protein (SEQ ID NO:2), and 94.8% identical tothe 7D_A06 protein (SEQ ID NO:8) (Table 3).

Two additional members of the 3D_C12 family were also identified. A genedesignated 3D_C12-10 (FIG. 3A-3G; coding sequence set forth as nt 1-1551of SEQ ID NO:3; predicted amino acid sequence set forth in SEQ ID NO:4)was recovered from an amplification reaction using a PCR primercomplementary to a sequence in the 3′ flanking region of 3D_C12 togetherwith a Bluescript II-specific primer (and the converter library astemplate). 3D_C12-10 differs at only five nucleotide positions from the3D_C12-7 nucleotide sequence (SEQ ID NO:5) (FIG. 3A-3G), and at only twoamino acids positions from the predicted 3D_C12-7 protein product (SEQID NO:6) (FIG. 4).

With the completion of the nonconverter EST database, another member ofthe 3D_C12 gene family was revealed. The partial DNA sequence of131A_A02 (SEQ ID NO:11; predicted amino acid sequence set forth in SEQID NO:12) that is found in this database is 98.0% identical to thecorresponding sequence of 3D_C12, and 94.0% identical to the same regionof 7D_A06 (FIG. 3A-3G and Table 2). As described in the previoussection, 131A_A02 is a member of the 3D_C12 gene family that wasrepresented on the comprehensive unigene chip used in microarray assaysas described elsewhere herein.

EXAMPLE 5 Transgenic Plant Analysis of Members of the 3D_C12 Gene Family

To determine whether members of the 3D_C12 family of cytochrome P450genes are involved in the metabolic conversion of nicotine tonornicotine, transgenic plants were generated using constructs designedto either enhance or inhibit gene expression. To test the effects ofdown-regulating gene activity, an RNA interference (RNAi) strategy wasemployed. A 99-bp region of 3D_C12 located immediately upstream of thestop codon (FIG. 3A-3G), was used to create a construct that would forma dsRNA hairpin within the plant cell. Such dsRNA structures are knownto activate an RNAi silencing complex that leads to the degradation ofboth transgene RNAs and endogenous RNAs that are identical or highlyhomologous to the sequence found in the dsRNA (Wesley et al. (2001)Plant J. 27: 581-590; Waterhouse & Helliwell (2002) Nat. Gen. Rev. 4:29-38).

Given that each member of the 3D_C12 characterized as described hereinshares over 90% DNA sequence identity, an RNAi construct synthesizedagainst one member was expected to silence the entire gene family.Specifically, the RNAi construct generated against the 3D_C12 sequenceshares sequence identities of 90/99 and 91/99 with the 7D_A06 and3D_C12-7 cDNAs, respectively, over this region (FIG. 3A-3G). The3D_C12/RNAi construct (also referred to in Example 7 as the 3D_C12Ri99construct) was cloned downstream of the constitutive 35S promoter ofcauliflower mosiac virus (CaMV) and introduced into the strong converterBurley tobacco line DH 98-325-6 using Agrobacterium-mediatedtransformation.

A hallmark of RNAi-mediated silencing is the marked reduction insteady-state transcript accumulation of the gene whose activity has beendown-regulated. To confirm that gene silencing of the 3D_C12 gene familyhad occurred in the plants showing low nornicotine phenotypes, aNorthern blot analysis was conducted using RNAs isolated from three ofthe transgenic plants possessing 3D_C12/RNAi constructs and displayinglow nornicotine phenotypes, two individuals transformed with the3D_C12/RNAi construct yet still showing high levels of nornicotine, andone of the vector-only control plants.

To assess the affects of overexpression of gene activity, the cDNAs fromthe three members of the 3D_C12 gene family for which we first obtainedfull-length sequence information (3D_C12, 7D_A06, and 3D_C12-7) werecloned in their sense orientations downstream of the 35S CaMV promoter.These constructs were subsequently introduced into N. tabacum cultivarPetite Havana using Agrobacterium-mediated transformation. The PetiteHavana line is commonly used by researchers because of its shorterstature and abbreviated generation time in relation to commercialtobacco cultivars. The converter/nonconverter status of the PetiteHavana cultivar is unknown, but the alkaloid assays of the presentapplication clearly showed that the plants in our possession were strongconverters.

Although the host plants in these experiments were converters, thepresent strategy was to conduct alkaloid assays on green, non-curedtissue, where minimal nornicotine accumulates in converter andnonconverter plants alike (and the 35S CaMV promoter is very active). Infact, a nonconverter line was purposely chosen because tissue culturing,as required when conducting Agrobacterium-mediated transformation, isknown to enhance the frequency of genetic conversion and would thuspotentially complicate interpretation of results (e.g., assessingwhether a novel phenotype was solely attributable to the transgene asopposed to being the result of the plant having undergone geneticconversion).

Results

Given the high degree of variability typically observed amongindependent transgenic plants transformed with the same transgeneconstruct, 10 independently transformed individuals were selected toassess the effects of the 3D_C12/RNAi construct on the metabolicconversion of nicotine to nornicotine. Leaves from each of the 103D_C12/RNAi individuals, in addition to two control plants transformedwith the pBI121 vector alone, were treated with ethephon and cured forseven days. Alkaloid analysis of these materials is shown in Table 4.

TABLE 4 Alkaloid analysis of DH 98-325-6 plants independentlytransformed with the 3D_C12/RNAi construct (and pBI121 vector control).Leaves were treated with ethephon and cured for seven days. % % % %Nico- Nor- Anab- % Conver- Sample tine* nicotine* asine* Anatabine*sion** 3D_C12 RNAi (1) 3.149 0.100 0.012 0.159 2.8 3D_C12 RNAi (2) 2.5690.193 0.009 0.110 7.0 3D_C12 RNAi (3) 2.175 0.064 0.007 0.080 2.9 3D_C12RNAi (4) 3.517 0.125 0.012 0.139 3.4 3D_C12 RNAi (5) 1.085 0.868 0.0090.119 44.4 3D_C12 RNAi (6) 0.025 2.260 0.011 0.122 98.9 3D_C12 RNAi (7)0.027 1.867 0.011 0.122 98.6 3D_C12 RNAi (8) 2.268 0.128 0.009 0.102 5.33D_C12 RNAi (9) 2.197 0.133 0.008 0.099 5.7 3D_C12 RNAi (10) 2.434 0.1120.009 0.110 4.4 vector control (3) 1.811 1.1735 0.018 0.170 48.9 vectorcontrol (11) 0.290 2.090 0.013 0.143 87.8 *percentage of leaf dry weight**[% nornicotine/(% nicotine + nornicotine)] × 100

Typical of line DH 98-325-6, ethephon treatment and curing resulted insubstantial nornicotine production in the two control plants (48.9% and87.8% conversion of nicotine to nornicotine). In dramatic contrast,seven of the ten independent transgenic plants possessing the3D_C12/RNAi construct displayed minimal nicotine to nornicotineconversion, with conversion percentages ranging from 2.8 to 7.0 percent.The other three 3D_C12/RNAi lines displayed alkaloid contents similar tothe vector-only control plants. Concentrations of the minor alkaloidsanabasine and anatabine did not appear to be significantly influenced bythe presence or absence of the 3D_C12/RNAi transgene (Table 4).

Although the cDNA insert of the 3D_C12-7 gene was used as the specifichybridization probe, at the hybridization and wash conditions used inthis experiment, cross-hybridization to the entire 3D_C12 gene familywould be expected. As shown in FIG. 5, a strong hybridization signal wasdetected in each plant showing a high nornicotine phenotype, and minimalhybridization was detected in the plants transformed with the3D_C12/RNAi construct that showed a low nornicotine phenotype. We thusconclude that the effective silencing of the 3D_C12 gene family inhibitsthe metabolic conversion of nicotine to nornicotine in tobacco.

Alkaloid analysis of the Petite Havana transgenic plants is shown inTable 5. Four independently transformed plants containing the 35S:3D_C12and 35S:3D_C12-7 constructs were tested along with seven independent35S:7D_A06 individuals and three plants independently transformed withthe pBI121 control vector. As expected, the green, non-cured leaves ofthe three vector-only control plants contained minimal amounts ofnornicotine. Likewise, all plants transformed with the 35S:3D_C12 and35S:7D_A06 constructs showed minimal metabolic conversion of nicotine tonornicotine. A very different phenotype, however, was observed withplants transformed with 35S:3D_C12-7. All four plants independentlytransformed with this construct contained nornicotine as the predominantalkaloid in the green, nontreated leaf; nicotine to nornicotineconversion percentages ranged from 94.6 to 98.6.

TABLE 5 Alkaloid analysis of individual Petite Havana plants transformedwith 3D_C12, 3D_C12-7, 7D_A06 constructs or the pBI121 vector control.Green leaves were harvested and analyzed without treatment or curing. %% % % Nico- Nor- Anab- % Conver- Sample tine* nicotine* asine*Anatabine* sion** vector control (2) 0.673 0.018 0.006 0.018 2.6 vectorcontrol (8) 0.605 0.014 0.005 0.016 2.3 vector control (10) 0.694 0.0170.004 0.018 2.4 35S:3D_C12 (1) 0.706 0.005 0.006 0.020 0.7 35S:3D_C12(2) 0.814 0.022 0.007 0.017 2.6 35S:3D_C12 (3) 0.630 0.010 0.003 0.0121.6 35S:3D_C12 (4) 0.647 0.010 0.004 0.011 1.5 35S:3D_C12-7 (1) 0.0050.347 0.002 0.012 98.6 35S:3D_C12-7 (2) 0.006 0.255 0.002 0.009 97.435S:3D_C12-7 (3) 0.017 0.300 0.002 0.010 94.6 35S:3D_C12-7 (4) 0.0100.384 0.002 0.015 97.5 35S:7D_A06 (1) 0.761 0.011 0.005 0.018 1.435S:7D_A06 (2) 0.507 0.009 0.003 0.007 1.7 35S:7D_A06 (4) 0.653 0.0150.006 0.015 2.2 35S:7D_A06 (5) 0.643 0.013 0.004 0.018 2.0 35S:7D_A06(6) 0.521 0.007 0.004 0.014 1.3 35S:7D_A06 (7) 0.716 0.015 0.005 0.0202.1 35S:7D_A06 (8) 0.701 0.027 0.004 0.018 3.7 *percentage of leaf dryweight **[% nornicotine/(% nicotine + nornicotine)] × 100

EXAMPLE 6 Cosuppression of the 3D_C12 Gene Family

In addition to the major conclusion that the 3D_C12-7 gene was capableof mediating nicotine to nornicotine conversion, one additionalobservation stood out in the alkaloid analyses of the Petite Havanatransgenic plants. The alkaloid results reported in Table 5 togetherwith additional alkaloid assays conducted independently (data not shown)consistently showed one of the plants transformed with the 35S:3D_C12construct (35S:3D_C12(1)) as having less nornicotine in the green,nontreated leaf than any other plant in this study. This may be theresult of cosuppression of the 3D_C12 gene family in this specificplant, a phenomenon frequently observed in transgenic plants even when atransgene is expressed in its sense orientation (Fagard and Vaucheret(2000) Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 167-194).

If plant 35S:3D_C12 (1) was truly displaying a cosuppression phenotype,this phenotype would be expected to be maintained even upon ethephontreatment and curing of the leaves, similar to the low nornicotinephenotypes conferred by the 3D_C12/RNAi construct in the convertergenotype DH 98-325-6 as described above. To test this prediction,alkaloid profiles were determined on ethephon treated, cured leaves of35S:3D_C12 (1) and two vector-only control plants. As shown in Table 6,ethephon treatment and curing resulted in over 97% nicotine tonornicotine conversion in the two control plants whereas similarlytreated 35S:3D_C12 (1) leaves displayed negligible conversion (0.6%).Leaves from five other plants expressing either 35S:3D_C12 and35S:7D_A06 transgenes were also subjected to ethephon treatment andcuring. In each case a high nornicotine phenotype was observed, similarto the vector-only control plants (data not shown).

TABLE 6 Alkaloid analysis of 35S:3D_C12 (1) and pBI121 vector controlsplants. Leaves were treated with ethephon cured for seven days. % % % %Nico- Nor- Anab- % Conver- Sample tine* nicotine* asine* Anatabine*sion** vector control (8) 0.009 0.425 n.d. 0.011 97.9 vector control(10) 0.008 0.560 n.d. 0.025 98.6 35S:3D_C12 (1) 1.185 0.007 n.d. 0.0200.6 *percentage of leaf dry weight **[% nornicotine/(% nicotine +nornicotine)] × 100 n.d., not detected

Finally, Northern blot assays were conducted on select plantsrepresenting each of the Petite Havana transgenic genotypes (FIG. 6).Using a 3D_C12-7 cDNA as a hybridization probe, minimal signal wasdetected with RNAs isolated from green, nontreated leaves of thevector-only control plant. In contrast, hybridization was easilydetected in RNA samples from all four independent transgenic plantspossessing the 35S:3D_C12-7 construct. A strong hybridization signal wassimilarly observed using RNAs from all other transgenic plants testedthat were transformed with the 35S:3D_C12 and 35S:7D_A06 constructs,with the exception of the low nornicotine containing plant 35S:3D_C12(1).

Overall results of the Northern blotting assays show that the 35S CaMVpromoter was generally effective in mediating a high level of geneexpression for each of the three members of the 3D_C12 gene familytested in this study. Failure to detect a hybridization signal in plant35S:3D_C12 (1) is consistent with the interpretation that the 3D_C12gene family has been silenced via cosuppression in this individual.

EXAMPLE 7 Additional Characterization and Suppression of Additional3D_C12 Genes

A second RNAi construct was prepared using polynucleotide sequences fromthe 3D_C12-7 sequence. The assembly of the 3D_C12-7/RNAi expressioncassette followed the same basic steps as those outlined for 3D_C12/RNAiabove. Briefly, a 298-bp sense and antisense strand of the 3D_C12-7 cDNA(SEQ ID NO:5) corresponding to the region between nucleotide positions297 and 594 of the coding sequence (positions 1-1551 of SEQ ID NO:5)were ligated into the pKYLX801 vector downstream and upstream of the151-bp soybean omega-6 fatty acid desaturase intron (see GenBankAccession No. DQ672337), respectively. The primers (E4SFwd and E4SRev)used for the isolation of the 298-bp region by sense and antisense armswere 5′-AAGCTTTGACGCCATTTTTTCCAATCG-3′ (SEQ ID NO:27), and5′-CTCGAGTTTTCCAGCGATCATCTTCAC-3′ (SEQ ID NO:28), respectively. The RNAicassette was excised from pKYL801 and placed between a strong CaMV35S²promoter and a rubisco small subunit terminator of the binary plantexpression vector, pKYLX71 (see FIG. 8). In the discussions below, thisRNAi construct is referred to as the 3D_C12-7-Ri298 construct.

Transgenic tobacco plants were generated via Agrobacterium-mediatedtransformation following the procedures provided above. Briefly,transformed burley tobacco plants were regenerated from calli onMurashige-Skoog (MS) medium supplemented with 100 mg/L kanamycin andplant hormones in a growth room maintained at 25° C. under a 16 hr/8 hrlight/dark cycle. Calli were transferred to fresh selection media every2-3 weeks until shoots appeared. Small shoots were transferred torooting media to allow root development for 2 weeks. Fully regeneratedplants were transferred to a greenhouse and grown under standardconditions.

SYBR® Green I Chemistry

Total RNA was isolated from cured leaves of converter and nonconverterburley tobacco plants using the TRIzol® reagent (Invitrogen, LifeTechnologies, Carlsbad, Calif.). Purified RNA was treated withRNase-free DNase (TURBO DNA-free™, Ambion, Austin, Tex.). First strandcDNA was synthesized using 5 μg of total RNA and the StrataScript®First-Strand Synthesis System (Stratagene, Cedar Creek, Tex.). Relativequantitative RT-PCR was employed for determining the abundance of the3D_C12-7 cDNA using SYBR® Green I fluorescence chemistry Morrison et al.(1998) Biotechniques 24:954-962.

A calibration curve was generated with a serial dilution of the 3D_C12-7cDNA cloned into the pGEM®-T Easy vector (Promega Corporation, Madison,Wis.). The RT-PCR mixture contained 2.5 mM MgCl₂, 125 μM each dNTP, 0.5μM each primer, 0.5×SYBR® Green I, 0.5 μg cDNA (or 1 μl referenceplasmid), and 1.25 U Platinum Taq polymerase (lnvitrogen LifeTechnologies). The sequences of the allele-specific 3D_C12-7 primers(E4SyFwd and E4SyRev) were 5′-ACGTGATCCTAAACTCTGGTCTG-3′ (E4SyFwd (SEQID NO:29)) and 5′-GCCTGCACCTTCCTTCATG-3′ (E4SyRev (SEQ ID NO:30)).RT-PCR was performed in a BioRad iCycler thermocycler (BioRadLaboratories, Hercules, Calif.) set to the following protocol: 95° C.for 2 min; 35 cycles of 95° C. for 30 sec, 55° C. for 30 sec 72° C. for50 sec, followed by final extension at 72° C. for 5 min. A 165-bpfragment of the α-tubulin gene was used as an internal standard.3D_C12-7 cDNA concentration was determined from the transcript-specificcalibration curve and normalized to the internal standard.Fold-induction was calculated by dividing the normalized fluorescencevalues of the converter by the nonconverter samples. Melting-curveanalysis was used to confirm the purity of PCR products as described inRirie et al. (1997) Anal. Biochem. 245:154-160. Two plants were sampledper treatment and amplifications were repeated three times.

TaqMan® Chemistry

Total RNA was isolated from tobacco lines using TRIzol reagent. PurifiedRNA was treated with RNase-free DNase (TURBO DNA-free™). First strandcDNA was synthesized using 10 μg of total RNA and the High Capacity cDNAArchive Kit (Applied Biosystems, Foster City, Calif.). The RT-PCRmixture contained 1× TaqMan® Universal PCR Master Mix (AppliedBiosystems, Foster City, Calif.), 400 nM of each primer (E4TmFwd andE4TmRev), 250 nM TaqMan® minor groove binder (MGB) probe (E4MGB), 2 ngof cDNA, and nuclease-free water (Afonina et al. (2002) Biotechniques32:940-949). The primer and probe sequences were5′CGGTAATCGGCCATCTTTTC-3′ (E4TmFwd (SEQ ID NO:31)),5′-CCGAGTTTTCGAGCTAATGGA-3′ (E4TmRev(SEQ ID NO:32)), and5′-CAATGACGAACGGCGACAG-3′ (MGB probe(SEQ ID NO:33)). RT-PCR wasperformed in an ABI 7500 Real-Time System (Applied Biosystems, FosterCity, Calif.) set to the following protocol: 50° C. for 2 min; 95° C.for 10 min; 40 cycles of 95° C. for 15 sec, 60° C. for 1 min.Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as theendogenous control to normalize the amount of cDNA template in thereactions. Fold-change was determined by dividing the normalizedfluorescence values of each sample by those obtained from a nonconverteror uncured control sample. For each treatment, RNA was isolated fromthree independent plants and amplifications were repeated 3 times perRNA sample.

Northern and Southern Blot Analyses

Total RNA was isolated from cured tobacco leaves using the TRIzolreagent according to the manufacturer's instructions (Invitrogen, LifeTechnologies). Total RNA samples were separated on 1.2% TBE agarose gel,and transferred to positively charged nylon membranes by electroblottingwith 2×TBE buffer. Membranes were UV crosslinked and washed in 2×SSC for5 min. Northern blot hybridization, washing, and detection were carriedout using the digoxigenin (DIG) System as described by the manufacturer(Roche Diagnostics Corp., Indianapolis, Ind.). The 1.8 kb full-lengthORF of the 3D_C12-7 cDNA was labeled with DIG and used as a probe.

Genomic DNA was extracted with DNAzol® (Invitrogen, Life Technologies)from green tobacco leaves according to manufacturer's protocol. Afterincubation with FcoRl or Ncol restriction enzymes overnight, 15 μg ofthe digested DNA was separated on 0.7% TBE agarose gel, depurinated with0.25 M HCl for 10 min, and denaturated with 0.5 N NaOH for 30 minutes.DNA was blotted overnight by capillary transfer onto positively chargednylon membranes (Roche Diagnostics Corp.) and hybridized at 65° C.overnight with a 515-bp DIG-labeled fragment of the neomycinphosphotransferase II (NPT II) gene. Hybridization, washing, anddetection were performed according to the protocols supplied with theDIG System. The primers used for the amplifications of the Northern andSouthern hybridization probes were

E4FIFwsd (5′-ATGGTTTTTCCCATAGAAGCC-3′ (SEQ ID   NO: 34)),E4FIRev (5′-TTTTTGGGACAATCAGTCAAT-3′ (SEQ ID NO: 35)),KanFwd (5′-TGAATGAACTGCAGGACGAG-3′ (SEQ ID NO: 36)), andKanRev (5′-AATATCACGGGTAGCCAACG-3′ (SEQ ID NO: 37)).

Alkaloid Analysis

Tobacco leaves were harvested and air dried in an oven at 50° C. for 2days. A 100 mg sample of crushed, dried leaf is added to 0.5 ml of 2 NNaOH in a 20 ml scintillation vial. The sample was mixed and allowed toincubate for 15 minutes at room temperature. Alkaloids were extracted bythe addition of 5 ml of extraction solution [0.04% quinoline (wt/vol)dissolved in methyl-t-butyl ether] and gently rotated on a linear shakerfor 3 hours. Following phase separation, an aliquot of the organic phasewas transferred to a sample vial. Samples were analyzed using aPerkinElmer Autosystem XL (PerkinElmer, Boston, Mass.) gas chromatographequipped with a flame ionization detector, a 4 mm split/splitless glassliner and a 30 m×0.53 mm ID DB-5 column. Chromatographic conditions wereas follows: detector temperature: 250° C.; injector temperature: 250°C.; helium flow at 120° C.: 20 ml/min; injection volume: 2 μ1; columnconditions: 120°, hold 1 min, 20-280° C. at 30° C. /min ramping rate,hold at 280° C. for 2 min. Alkaloid composition was determined by theTotalChrome Navigator software using a calibration curve. Means of thealkaloid measurements were separated according to Fisher's Protected LSD(PROC MIXED).

Plants

Double haploid burley tobacco lines DH 98-325-5 (325-5; nonconverter)and DH98-325-6 (325-6; converter) described above were used in allexperiments, except for the fluorogenic 5′ nuclease (TaqMan®)chemistry-based RT-PCR assays where the isogenic DH 91-1307-46(nonconverter) and DH 91-1307-46 (converter) lines were used. All plantswere grown in a controlled environment greenhouse equipped withsupplemental lighting providing, a 14 hr/10 hr light/dark cycle.

For curing, tobacco leaves were collected from converter andnonconverter plants about 1 month before flowering and treated bydipping each leaf twice for 10 sec, into 2% ethephon and dried for 2hours. Leaves were cured for up to two weeks in plastic bags, under darkconditions, until they turned yellow. Cured leaves were used for theNorthern and alkaloid analyses. Samples of cured leaves subjected to GCanalysis were dried at 50° C. for 2 days. For Southern analysis, greentobacco leaves of adult plants were used. To produce T₁ generationtransgenic plants, primary transformants (T₀) were self-pollinated, andthe harvested T₁ seed was screened by germinating seedlings on MS-agarplates containing 100 mg/L kanamycin for 6 weeks. Survivors weretransplanted to soil and grown in a greenhouse as described above.Plants were fertilized with Peter's Professional All Purpose Plant Food(20-20-20; Spectrum Brands Inc., Madison, Wis.) once a week.

RT-PCR Analysis of 3D_C12-7 Expression in Converter and NonconverterTobacco

To further characterize the role of 3D_C12-7 in nicotineN-demethylation, experiments were performed to demonstrate that theregulation of 3D_C12-7 expression is consistent with the levels ofnicotine N-demethylation activity observed in converter versusnonconverter tobacco.

To determine the rate of 3D_C12-7 mRNA accumulation in converter andnonconverter tobacco, an allele-specific real-time RT-PCR strategy wasemployed. Because RT-PCR involves the detection and measurement of theamplification products of a PCR template, the use of allele-specificprimers allows the quantification of a single isoform among a group ofhighly homologous sequences. For accurate quantification of the 3D_C12-7transcript, two different segments of the 3D_C12-7 coding region wereamplified and both SYBR® Green and TaqMan® chemistries were used togenerate fluorescence signals. RT-PCR analysis using the SYBR® Green Ichemistry revealed an 80-fold increase in the levels of the 3D_C12-7transcript in the cured leaves of converter versus nonconverter tobacco.A single peak melting curve and gel electrophoretic analyses of theamplicons confirmed the homogeneity of the PCR products.

In the TaqMan® chemistry-based RT-PCR experiment, 3D_C12-7 transcriptlevels were quantified in untreated and ethephon-treated converter andnonconverter tobacco leaves that were cured for 0, 1 or 5 days. Lowlevels of 3D_C12-7 transcripts were detected in the uncured leaves orfollowing a 1-day curing period regardless of conversion type orethephon treatment. Similarly, base line levels of 3D_C12-7transcription were observed in converter or nonconverter leaves thatwere cured for 5 days without ethephon treatment. In contrast, a7.5-fold increase in 3D_C12-7 transcript accumulation was detected inthe cured leaves of converter versus nonconverter tobacco, and a 70-foldincrease was observed in the uncured versus cured leaves of a convertertobacco variety when ethephon treatment preceded the 5-day curingperiod. While not intending to be limited by any particular theory,these results suggest that 3D_C12-7 is a major contributor to nicotineN-demethylation and is strongly inducible by ethylene in senescingtobacco leaves.

Suppression of Nicotine to Nornicotine Conversion by the 3D_C12-Ri99 and3D C12-7-Ri298 Constructs

To compare the extent to which 3D_C12 and 3D_C12-7 mediate thesuppression of nornicotine production, converter and nonconverter burleytobacco plants were transformed with the two gene silencing vectors. Ten(10) transgenic plants were regenerated per RNAi construct. About 80% oftobacco plants overexpressing either the 99-bp or 298-bp inverted repeatshowed reduced nornicotine levels compared to the empty vector controls(Tables 7 and 8). In the nonconverter genotype, 3D_C12-Ri99 and3D_C12-7-Ri298 expression reduced nicotine to nornicotine conversion byabout 1.8-fold (2.0%) and 3.0-fold (1.2%), respectively, in comparisonto the rate of conversion detected in the vector controls (3.6%) (Table7). Among the silenced nonconverter plants, the lowest conversion levelof 0.9% was achieved using the 3D_C12-7-Ri298 construct (Table 7).

TABLE 7 Alkaloid analysis of nonconvertor burley tobacco plantstransformed with the 3D_C12-Ri99 or 3D_C12-7-Ri298 construct. Line^(c) %Nicotine^(d) % Nornicotine^(d) % Conversion^(e) 3D-C12-Ri99 1 1.6930.034 2.0 2 1.435 0.031 2.1 3 2.095 0.043 2.0 4 2.868 0.053 1.8 5 0.9470.025 2.6 6 2.357 0.043 1.8 7 2.599 0.043 1.6 8 0.796 0.020 2.4 9 2.1780.039 1.8 10  3.162 0.061 1.9 MEAN 2.013 0.039 2.0a STE 0.748 0.012 0.33D_C12-7-Ri298 3 1.806 0.020 1.1 4 1.948 0.207 1.4 5 2.061 0.020 1.0 62.704 0.040 1.5 8 2.652 0.023 0.9 9 1.074 0.015 1.3 MEAN 2.041 0.0241.2b STE 0.550 0.008 0.2 Vector Control^(g) 1 1.206 0.052 4.2 2 1.2650.038 2.9 3 1.752 0.058 3.2 4 1.230 0.072 5.6 5 1.777 0.060 3.3 6 1.5360.044 2.8 MEAN 1.461 0.054 3.6^(c) STE 0.240 0.011 1.0 aTobacco leaveswere treated with ethephon and cured for 2 weeks at 25° C. bOf theplants transformed with an RNAi construct, only silenced individuals areshown. Alkaloid data represent the means of 2 measurements. ^(c)Numbersrepresent independently transformed individuals. ^(d)Percentage of leafdry weight. ^(e)[% nornicotine/(% nicotine + % nornicotine)] × 100;values followed by different letters are significantly differentaccording to Fisher's Protected LSD (0.05). ^(f)STE, standard error^(g)Tobacco plants transformed with only pKYLX71 vector were used ascontrols.

Relative to nonconverter tobacco, nornicotine accumulation wassuppressed even more dramatically in the silenced individuals of thestrong converter plants (Table 8). Using 3D_C12-Ri99 constructs,nicotine conversion was reduced to levels as low as 4.5% in 3D_C12-Ri99-transformed 325-6 tobacco plants in sharp contrast to the 325-6control plants exhibiting about 98% conversion rates; Table 8). However,using the 3D_C12-7-Ri298 construct even greater reductions in nicotineconversion were obtained (Table 8). Four 3D_C12-7-Ri298-transformedindividuals converted as low as 0.8% of their nicotine to nornicotine,and the arithmetic mean across the 9 silenced transformants was 0.9%conversion. All silenced plants were morphologically indistinguishablefrom both the empty vector and wild-type controls (data not shown).

TABLE 8 Alkaloid analysis of convertor burley tobacco plants transformedwith the 3D_C12-Ri99 or 3D_C12-7-Ri298 construct. Line^(c) %Nicotine^(d) % Nornicotine^(d) % Conversion^(e) 3D_C12-Ri99 1 3.4190.100 2.8 2 2.569 0.193 7.0 3 2.175 0.064 2.9 4 3.517 0.125 3.4 8 2.2680.128 5.3 9 2.197 0.133 5.7 10  2.434 0.112 4.4 MEAN 2.654 0.122 4.5aSTE^(g) 0.573 0.039 1.6 3D_C12-7-Ri298^(b) 1 2.043 0.020 1.0 2 3.4270.026 0.8 3 2.603 0.020 0.8 5 2.427 0.030 1.2 6 2.106 0.021 1.0 7 1.4120.015 1.1 8 3.328 0.028 0.8 9 1.493 0.015 1.0 10  2.065 0.018 0.8 MEAN2.323 0.021 0.9^(b) STE 0.669 0.005 0.1 Vector Control^(h,i) 1 0.1261.550 92.5 2 0.330 2.604 88.8 3 0.060 1.419 95.9 4 0.114 1.267 91.7 50.119 1.303 91.6 MEAN 0.150 1.628 92.1^(c) STE 0.093 0.498 2.3 aTobaccoleaves were treated with ethephon and cured for 2 weeks at 25° C. ^(b)Ofthe plants transformed with an RNAi construct, only silenced individualsare shown. ^(c)Numbers represent independently transformed individuals.^(d)Percentage of leaf dry weight. ^(e)[% nornicotine/(% nicotine + %nornicotine)] × 100; values followed by different letters aresignificantly different according to Fisher's Protected LSD (0.05).^(f)STE, standard error ^(g)Tobacco plants transformed with only pKYLX71vector were used as controls.

To test the heritability of nornicotine suppression in the3D_C12-7-Ri298-transformed plants, a set of 3D_C12-7-Ri298-transformedconverter and nonconverter lines that displayed the lowest levels ofnicotine conversion were advanced to the T₁ generation (Table 9).Because segregation of the transgene(s) occurs in the T₁ progeny,transgenic individuals were identified by selecting seedlings capable ofgrowing on kanamycin-containing media. Nine kanamycin-resistantprogenies of each selected T₀-generation 3D_C12-7-Ri298 transformant andfour kanamycin-resistant individuals from each selected vector controlline were analyzed for alkaloid content. The rate of nicotine conversiondid not differ significantly between the primary 3D_C12-7-Ri298transformants and their T₁ progeny, indicating high heritability of thenornicotine suppression trait (see Tables 7, 8, and 9). However,advancing the “nonconverter” vector control line by a single generationincreased the nicotine to nornicotine conversion rate from 4.2% to anaverage value of 11.6%, illustrating the high degree of instability ofthe conversion locus in transgenic plants lacking the3D_C12-7-Ri298-specific RNAi construct (Tables 7 and 9). Overall, theseresults show that RNAi-mediated silencing of the 3D_C12 gene subfamilyis a highly effective means of lowering nornicotine production in bothnonconverter and strong converter tobacco plants.

TABLE 9 Alkaloid analysis of T₁- generation 3D_C12-7-Ri298transformants. Line % Nicotine^(c) % Nornicotine^(c) % Conversion^(d)DH98-325-5 (nonconverter) 3D_C12-7-Ri298#3 Mean 1.764 0.024 1.4a STE0.456 0.004 0.3 3D_C12-7-Ri298#5 Mean 1.500 0.020 1.3a STE 0.306 0.0060.3 3D_C12-7-Ri298#8 Mean 1.772 0.020 1.2a STE 0.409 0.003 0.3 VectorControl#1^(e) Mean 1.466 0.203 11.6b STE 0.713 0.161 9.7 DH98-325-6(converter) 3D_C12-7-Ri298#2 Mean 1.970 0.019 1.0a STE 0.536 0.004 0.33D_C12-7-Ri298#8 Mean 1.623 0.022 1.3a STE 0.300 0.002 0.23D_C12-7-Ri298#10 Mean 1.419 0.017 1.3a STE 0.515 0.004 0.3 VectorControl#2^(e) Mean 0.028 1.170 97.6^(c) STE 0.006 0.234 0.5 aTobaccoleaves were treated with ethephon and cured for 2 weeks at 25° C. bMeansand standard errors (STE) represent 9 and 4 T₁ progenies of the3D_C12-7-Ri298 construct and empty vector-transformed (vector control)lines, respectively. ^(c)Percentage of leaf dry weight. ^(d)[%nornicotine/(% nicotine + % nornicotine)] × 100; values followed bydifferent letters are significantly different according to Fisher'sProtected LSD (0.015). ^(e)Tobacco plants transformed with only pKYLX71vector were used as controls.

Furthermore, transforming tobacco with the 3D_C12-7-298 constructconferred a 3.6-fold reduction in nicotine conversion relative totypical nonconverter control plants without affecting plant growth anddevelopment.

To demonstrate that the down-regulation of nornicotine production in3D_C12-7-298-transformed tobacco was concomitant with a reduction of the3D_C12 gene subfamily transcripts, a 3D_C12-7 cDNA probe was hybridizedto the total RNA isolated from cured leaves of nonconverter andconverter plants. A weak hybridization signal was generated by the RNAisolated from 3D_C12-7-Ri298 transformants displaying low nornicotinecontent in contrast to the strong signal produced by the RNA extractedfrom plasmid control or wild-type plants. These results indicate thatthe down-regulation of nicotine conversion was a result of RNAi-mediatedgene silencing of the nicotine N-demethylase gene(s).

Determination of Transgene Copy Number

To determine whether the integration of multiple 3D_C12-7-Ri298 copieswere required for producing transplants displaying very low nicotineN-demethylase activity, Southern analysis was performed on selectedindividuals exhibiting <1.5% nornicotine accumulation. The number oftransgenes varied widely among these plants including individualscontaining 1 copy (325-5, lines 5 & 8; 325-6, lines 2 & 8), 5 copies(325-6, line 10), and 6 copies (325-5, line 6) of the 3D_C12-7-Ri298construct using Southern blot analysis of genomic DNA digested with theEcoRI restriction enzyme. Transgene copy number was confirmed using NcoIdigested DNA (data not shown). These results indicate that theintegration of a single 3D_C12-7-Ri298 construct into the genome of astrong converter tobacco is sufficient for suppressing nornicotineproduction to very low levels.

General Conclusions

The analyses outlined in Examples 1-6 above resulted in the discovery ofa closely related P450 gene family, designated the 3D_C12 family, whosecollective steady-state transcript levels were significantly elevated inconverter tobacco plants that were actively metabolizing nicotine tonornicotine in comparison to their nonconverter counterparts. Transgenicplant analysis demonstrated that the suppression of gene expression ofthis P450 family in converter tobacco lines inhibited the metabolism ofnicotine to nornicotine to levels similar to that observed innonconverter plants. Furthermore, sense expression of severalindividuals of this closely related gene family identified one member,designated 3D_C12-7, as playing a direct role in the metabolicconversion of nicotine to nornicotine. Overexpression of 3D_C12-7 usinga strong constitutive promoter caused a dramatic increase in nornicotineproduction and accumulation in non-cured green leaves of transgenictobacco plants, a tissue where nicotine is normally the predominantalkaloid in converter and nonconverter plants alike. Given that thecytochrome P450 family member designated 3D_C12-10 differs from 3D_C12-7at only two amino acid residues immediately following the startmethionine and within the N-terminal signal sequence, it is predictedthat these encoded products function identically.

The contrast in alkaloid phenotypes between the 35S:3D_C12 (1) plant andvector-only control plants was most dramatic in leaves that had beenethephon treated and cured (0.6% conversion versus >97% conversion;Table 6). However, it is noteworthy that the nornicotine content of theco-suppressed 35S:3D_C12 (1) plant was reduced even in green, nontreatedleaves where the high nornicotine phenotype is typically not manifest inconverter or nonconverter tobacco lines. The green, nontreated leaves ofline 35S:3D_C12 (1) showed only 0.7% nicotine to nornicotine conversion,whereas every other plant in this experiment showed conversionpercentages ranging from 1.3 to 3.7 (Table 5). This result suggests thatthe inhibition of gene expression of the 3D_C12 family may prove to beeffective in the further lowering of nornicotine levels even in tobaccolines where genetic conversion isn't typically a major problem (such asflue-cured tobaccos) or in the nonconverter individuals in lines thatare prone to genetic conversion (such as Burley tobaccos).

Southern blotting assays using members of the 3D_C12 gene family ashybridization probes gives very complex banding patterns, suggestingthat more members of this gene family may exist even beyond those thathave been identified and characterized herein (data not shown). Thehypothesis that the 3D_C12 gene family is comprised of additionalmembers is further supported by the recent publication of 75 full-lengthtobacco P450 cDNAs of unknown function (U.S. Patent ApplicationPublication 20040162420). Within this list of P450s are additional cDNAsthat would, based on the work described herein, be placed within the3D_C12 family in view of their display of over 90% amino acid sequenceidentity to the protein sequences shown in FIG. 4.

With respect to the specific molecular function of the 3D_C12-7 gene orthe nearly identical family member 3D_C12-10, it is possible that itencodes the actual nicotine demethylase enzyme which catalyzes theoxidative N-demethylation of nicotine to nornicotine (FIG. 1).Alternatively, the 3D_C12-7 encoded enzyme or nearly identical 3D_C12-10encoded enzyme may produce a product that leads to the up-regulation ofthe nicotine demethylase activity of the leaf, as opposed to directlycatalyzing the N-demethylation reaction.

In addition, an allele-specific RT-PCR was employed to compare 3D-C12-7expression between converter and nonconverter plants (Example 7). Anapproximately 80-fold increase in 3D-C12-7 expression in converterversus nonconverter plants was identified using the SYBR®Green-chemistry RT-PCR assay. A 7.5-fold up-regulation was identified bythe TaqMan® chemistry-based RT-PCR experiment. While the DH 91-1307-46tobacco variety used in the TaqMan® chemistry-based RT-PCR experimentexhibits low to moderate levels of nicotine conversion, the DH98-325-5nonconverter plants used in the SYBR® Green-based RT-PCR assayconsistently convert a very low percentage of their nicotine tonornicotine. Expression of the 3D-C12-7 gene was induced at least 7-foldby ethylene in senescing leaves of converter tobacco plants.

An additional RNAi construct, 3D_C12-7-Ri298, was prepared based on aregion of the 3D_C12-7 polynucleotide that corresponds to nucleotidepositions 297 through 594 of SEQ ID NO:5. Expression of this RNAiconstruct allows for the suppression of nornicotine production in astrong converter tobacco line below the levels normally found innonconverter plants. The expression cassette of the 3D_C12-7-Ri298construct encoded an intron-spliced hairpin RNA in which the stem regionwas engineered from this 298-bp fragment of the 3D_C12-7 cDNA insertedas an inverted repeat. The loop of the hairpin was created by placing a151-bp intron of the FAD gene between the two sides of the palindromicsequences. An arm length of 298-bp was used for the inverted repeats.

3D_C12-7-Ri298-transformed plants accumulated less nornicotine thanthose harboring the 3D_C12-Ri99 construct (Tables 7 and 8). Nocorrelation was found between the number of copies of the 3D_C12-7-Ri298construct and nornicotine production (Tables 7 and 8). The3D_C12-7-Ri298 expression cassette enabled the production of tobaccowith a conversion rate as low as 0.8%, which is below the 3-5% ratedetected in burley lines used by seed producers. Such dramatic reductionin nornicotine production by targeting this particular region of the3D_C12-7 polynucleotide is an unexpected result. Also, suppression ofnornicotine production showed a high degree of heritability in the T₁progeny of the primary transformants (Table 9). Suppression ofnornicotine production in these transgenic plants yielded no obviousdifferences in growth and development when compared to wild-type plants.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1-84. (canceled)
 85. Cured tobacco derived from a tobacco plantcomprising a stable mutation in a wild type allele of a nicotinedemethylase comprising a coding sequence having at least 98% identity toSEQ ID NO: 3, wherein said mutation reduces or eliminates the expressionor activity of said nicotine demethylase, wherein the amount ofnornicotine or N′-nitrosonornicotine in said tobacco plant is reduced bygreater than about 10% compared to a control tobacco plant comprisingsaid wild type allele, and wherein said tobacco plant is a Nicotianatabacum plant.
 86. The cured tobacco of claim 85, wherein the tobaccoplant is homozygous for said mutation.
 87. The cured tobacco of claim85, wherein the tobacco plant has a non-converter phenotype andcomprises less than 5% nicotine demethylation.
 88. The cured tobacco ofclaim 85, wherein the tobacco plant is selected from the groupconsisting of a Burley type, a dark type, a flue-cured type, and anOriental type.
 89. The cured tobacco of claim 85, wherein the mutationis selected from the group consisting of a point mutation, a deletion,and an insertion.
 90. The cured tobacco of claim 85, wherein the wildtype allele encodes SEQ ID NO:
 3. 91. The cured tobacco of claim 85,wherein the stable mutation in a wild type allele is a mutation in anynucleotide corresponding to nucleotides 265 to 625 of SEQ ID NO:
 3. 92.The cured tobacco of claim 85, wherein the stable mutation in a wildtype allele is a mutation in any nucleotide corresponding to nucleotides700 to 1250 of SEQ ID NO:
 3. 93. The cured tobacco of claim 85, whereinthe stable mutation in a wild type allele is a mutation in anynucleotide corresponding to nucleotides 1420 to 1551 of SEQ ID NO: 3.94. The cured tobacco of claim 85, wherein the amount of nornicotine orN′-nitrosonornicotine in the tobacco plant is reduced by greater thanabout 20% compared to a control tobacco plant comprising the wild typeallele.
 95. The cured tobacco of claim 85, wherein the amount ofnornicotine or N′-nitrosonornicotine in the tobacco plant is reduced bygreater than about 60% compared to a control tobacco plant comprisingthe wild type allele.
 96. The cured tobacco of claim 85, wherein theamount of nornicotine or N′-nitrosonornicotine in the tobacco plant isreduced by greater than about 90% compared to a control tobacco plantcomprising the wild type allele.
 97. The cured tobacco of claim 85,wherein the cured tobacco material is flue-cured.
 98. The cured tobaccoof claim 85, wherein the cured tobacco material is air-cured.
 99. Thecured tobacco of claim 85, wherein the mutation inhibits production ofthe nicotine demethylase protein.
 100. The cured tobacco of claim 85,wherein the tobacco plant further comprises a stable mutation in a wildtype allele of a nicotine demethylase encoding a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NOs: 2,6, 8, 10, and
 12. 101. The cured tobacco of claim 85, wherein themutation is a null mutation.
 102. The cured tobacco of claim 85, whereinthe cured tobacco comprised cured tobacco leaf.